Novel Substituted Bicyclic Aromatic Compounds as S-Nitrosoglutathione Reductase Inhibitors

ABSTRACT

The present invention is directed to novel substituted bicyclic aromatic compounds useful as S-nitrosoglutathione reductase (GSNOR) inhibitors, pharmaceutical compositions comprising such compounds, and methods of making and using the same.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/991,973, filed Jun. 6, 2013. U.S. application Ser. No. 13/991,973 isa 35 U.S.C. §371 national phase application of PCT/US2011/065502, filedDec. 16, 2011 (WO 2012/083171). PCT/US2011/065502 claims priority toU.S. Provisional Application Ser. No. 61/423,799, filed Dec. 16, 2010.Each of these applications is incorporated hereby by reference in itsentirety.

FIELD OF THE INVENTION

The present invention is directed to novel substituted bicyclic aromaticcompounds, pharmaceutical compositions comprising such compounds, andmethods of making and using the same. These compounds are useful asinhibitors of S-nitrosoglutathione reductase (GSNOR).

BACKGROUND

The chemical compound nitric oxide is a gas with chemical formula NO. NOis one of the few gaseous signaling molecules known in biologicalsystems, and plays an important role in controlling various biologicalevents. For example, the endothelium uses NO to signal surroundingsmooth muscle in the walls of arterioles to relax, resulting invasodilation and increased blood flow to hypoxic tissues. NO is alsoinvolved in regulating smooth muscle proliferation, platelet function,and neurotransmission, and plays a role in host defense. Although NO ishighly reactive and has a lifetime of a few seconds, it can both diffusefreely across membranes and bind to many molecular targets. Theseattributes make NO an ideal signaling molecule capable of controllingbiological events between adjacent cells and within cells.

NO is a free radical gas, which makes it reactive and unstable, thus NOis short lived in vivo, having a half life of 3-5 seconds underphysiologic conditions. In the presence of oxygen, NO can combine withthiols to generate a biologically important class of stable NO adductscalled S-nitrosothiols (SNO's). This stable pool of NO has beenpostulated to act as a source of bioactive NO and as such appears to becritically important in health and disease, given the centrality of NOin cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA,89:7674-7677 (1992)). Protein SNO's play broad roles in the function ofcardiovascular, respiratory, metabolic, gastrointestinal, immune, andcentral nervous system (Foster et al., Trends in Molecular Medicine, 9(4):160-168, (2003)). One of the most studied SNO's in biologicalsystems is S-nitrosoglutathione (GSNO) (Gaston et al., Proc. Natl. Acad.Sci. USA 90:10957-10961 (1993)), an emerging key regulator in NOsignaling since it is an efficient trans-nitrosating agent and appearsto maintain an equilibrium with other S-nitrosated proteins (Liu et al.,Nature, 410:490-494 (2001)) within cells. Given this pivotal position inthe NO-SNO continuum, GSNO provides a therapeutically promising targetto consider when NO modulation is pharmacologically warranted.

In light of this understanding of GSNO as a key regulator of NOhomeostasis and cellular SNO levels, studies have focused on examiningendogenous production of GSNO and SNO proteins, which occurs downstreamfrom the production of the NO radical by the nitric oxide synthetase(NOS) enzymes. More recently there has been an increasing understandingof enzymatic catabolism of GSNO which has an important role in governingavailable concentrations of GSNO and consequently available NO andSNO's.

Central to this understanding of GSNO catabolism, researchers haverecently identified a highly conserved S-nitrosoglutathione reductase(GSNOR) (Jensen et al., Biochem J., 331:659-668 (1998); Liu et al.,(2001)). GSNOR is also known as glutathione-dependent formaldehydedehydrogenase (GSH-FDH), alcohol dehydrogenase 3 (ADH-3) (Uotila andKoivusalo, Coenzymes and Cofactors., D. Dolphin, ed. pp. 517-551 (NewYork, John Wiley & Sons, (1989)), and alcohol dehydrogenase 5 (ADH-5).Importantly GSNOR shows greater activity toward GSNO than othersubstrates (Jensen et al., (1998); Liu et al., (2001)) and appears tomediate important protein and peptide denitrosating activity inbacteria, plants, and animals. GSNOR appears to be the majorGSNO-metabolizing enzyme in eukaryotes (Liu et al., (2001)). Thus, GSNOcan accumulate in biological compartments where GSNOR activity is low orabsent (e.g., airway lining fluid) (Gaston et al., (1993)).

Yeast deficient in GSNOR accumulate S-nitrosylated proteins which arenot substrates of the enzyme, which is strongly suggestive that GSNOexists in equilibrium with SNO-proteins (Liu et al., (2001)). Preciseenzymatic control over ambient levels of GSNO and thus SNO-proteinsraises the possibility that GSNO/GSNOR may play roles across a host ofphysiological and pathological functions including protection againstnitrosative stress wherein NO is produced in excess of physiologicneeds. Indeed, GSNO specifically has been implicated in physiologicprocesses ranging from the drive to breathe (Lipton et al., Nature,413:171-174 (2001)) to regulation of the cystic fibrosis transmembraneregulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001)),to regulation of vascular tone, thrombosis, and platelet function (deBelder et al., Cardiovasc Res.; 28(5):691-4 (1994)), Z. Kaposzta, etal., Circulation; 106(24): 3057-3062, (2002)) as well as host defense(de Jesus-Berrios et al., Curr. Biol., 13:1963-1968 (2003)). Otherstudies have found that GSNOR protects yeast cells against nitrosativestress both in vitro (Liu et al., (2001)) and in vivo (de Jesus-Berrioset al., (2003)).

Collectively, data suggest GSNO as a primary physiological ligand forthe enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizesGSNO and consequently reduces available SNO's and NO in biologicalsystems (Liu et al., (2001)), (Liu et al., Cell, 116(4), 617-628(2004)), and (Que et al., Science, 308, (5728):1618-1621 (2005)). Assuch, this enzyme plays a central role in regulating local and systemicbioactive NO. Since perturbations in NO bioavailability has been linkedto the pathogenesis of numerous disease states, including hypertension,atherosclerosis, thrombosis, asthma, gastrointestinal disorders,inflammation, and cancer, agents that regulate GSNOR activity arecandidate therapeutic agents for treating diseases associated with NOimbalance.

Nitric oxide (NO), S-nitrosoglutathione (GSNO), and S-nitrosoglutathionereductase (GSNOR) regulate normal lung physiology and contribute to lungpathophysiology. Under normal conditions, NO and GSNO maintain normallung physiology and function via their anti-inflammatory andbronchodilatory actions. Lowered levels of these mediators in pulmonarydiseases such as asthma, chronic obstructive pulmonary disease (COPD)may occur via up-regulation of GSNOR enzyme activity. These loweredlevels of NO and GSNO, and thus lowered anti-inflammatory capabilities,are key events that contribute to pulmonary diseases and which canpotentially be reversed via GSNOR inhibition.

S-nitrosoglutathione (GSNO) has been shown to promote repair and/orregeneration of mammalian organs, such as the heart (Lima et al., 2010),blood vessels (Lima et al., 2010) skin (Georgii et al., 2010), eye orocular structures (Haq et al., 2007) and liver (Prince et al., 2010).S-nitrosoglutathione reductase (GSNOR) is the major catabolic enzyme ofGSNO. Inhibition of GSNOR is thought to increase endogenous GSNO.

Inflammatory bowel diseases (IBD's), including Crohn's and ulcerativecolitis, are chronic inflammatory disorders of the gastrointestinal (GI)tract, in which NO, GSNO, and GSNOR can exert influences. Under normalconditions, NO and GSNO function to maintain normal intestinalphysiology via anti-inflammatory actions and maintenance of theintestinal epithelial cell barrier. In IBD, reduced levels of GSNO andNO are evident and likely occur via up-regulation of GSNOR activity. Thelowered levels of these mediators contribute to the pathophysiology ofIBD via disruption of the epithelial barrier via dysregulation ofproteins involved in maintaining epithelial tight junctions. Thisepithelial barrier dysfunction, with the ensuing entry ofmicro-organisms from the lumen, and the overall loweredanti-inflammatory capabilities in the presence of lowered NO and GSNO,are key events in IBD progression that can be potentially influenced bytargeting GSNOR.

Cell death is the crucial event leading to clinical manifestation ofhepatotoxicity from drugs, viruses and alcohol. Glutathione (GSH) is themost abundant redox molecule in cells and thus the most importantdeterminant of cellular redox status. Thiols in proteins undergo a widerange of reversible redox modifications during times of exposure toreactive oxygen and reactive nitrogen species, which can affect proteinactivity. The maintenance of hepatic GSH is a dynamic process achievedby a balance between rates of GSH synthesis, GSH and GSSG efflux, GSHreactions with reactive oxygen species and reactive nitrogen species andutilization by GSH peroxidase. Both GSNO and GSNOR play roles in theregulation of protein redox status by GSH.

Acetaminophen overdoses are the leading cause of acute liver failure(ALF) in the United States, Great Britain and most of Europe. More than100,000 calls to the U.S. Poison Control Centers, 56,000 emergency roomvisits, 2600 hospitalizations, nearly 500 deaths are attributed toacetaminophen in this country annually. Approximately, 60% recoverwithout needing a liver transplant, 9% are transplanted and 30% ofpatients succumb to the illness. The acetaminophen-related death rateexceeds by at least three-fold the number of deaths due to all otheridiosyncratic drug reactions combined (Lee, Hepatol Res 2008; 38 (Suppl.1):S3-S8).

Liver transplantation has become the primary treatment for patients withfulminant hepatic failure and end-stage chronic liver disease, as wellas certain metabolic liver diseases. Thus, the demand fortransplantation now greatly exceeds the availability of donor organs. Ithas been estimated that more than 18 000 patients are currentlyregistered with the United Network for Organ Sharing (UNOS) and that anadditional 9000 patients are added to the liver transplant waiting listeach year, yet less than 5000 cadaveric donors are available fortransplantation.

Currently, there is a great need in the art for diagnostics,prophylaxis, ameliorations, and treatments for medical conditionsrelating to increased NO synthesis and/or increased NO bioactivity. Inaddition, there is a significant need for novel compounds, compositions,and methods for preventing, ameliorating, or reversing otherNO-associated disorders. The present invention satisfies these needs.

SUMMARY

The present invention provides novel substituted bicyclic aromaticcompounds. These compounds are useful as S-nitrosoglutathione reductase(“GSNOR”) inhibitors. The invention encompasses pharmaceuticallyacceptable salts, stereoisomers, prodrugs, metabolites, and N-oxides ofthe described compounds. Also encompassed by the invention arepharmaceutical compositions comprising at least one compound of theinvention and at least one pharmaceutically acceptable carrier.

The compositions of the present invention can be prepared in anysuitable pharmaceutically acceptable dosage form.

The present invention provides a method for inhibiting GSNOR in asubject in need thereof. Such a method comprises administering atherapeutically effective amount of a pharmaceutical compositioncomprising at least one GSNOR inhibitor or a pharmaceutically acceptablesalt, stereoisomer, prodrug, metabolite or N-oxide thereof, incombination with at least one pharmaceutically acceptable carrier. TheGSNOR inhibitor can be a novel compound according to the invention, orit can be a known compound which previously was not known to be aninhibitor of GSNOR.

The present invention also provides a method of treating a disorderameliorated by NO donor therapy in a subject in need thereof. Such amethod comprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, orN-oxide thereof, in combination with at least one pharmaceuticallyacceptable carrier. The GSNOR inhibitor can be a novel compoundaccording to the invention, or it can be a known compound whichpreviously was not known to be an inhibitor of GSNOR.

The present invention also provides a method of treating a cellproliferative disorder in a subject in need thereof. Such a methodcomprises administering a therapeutically effective amount of apharmaceutical composition comprising at least one GSNOR inhibitor or apharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, orN-oxide thereof, in combination with at least one pharmaceuticallyacceptable carrier. The GSNOR inhibitor can be a novel compoundaccording to the invention, or it can be a known compound whichpreviously was not known to be an inhibitor of GSNOR.

The methods of the invention encompass administration with one or moresecondary active agents. Such administration can be sequential or in acombination composition.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publiclyavailable publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control.

Both the foregoing summary and the following detailed description areexemplary and explanatory and are intended to provide further details ofthe compositions and methods as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description.

DETAILED DESCRIPTION

A. Overview of the Invention

Until recently, S-nitrosoglutathione reductase (GSNOR) was known tooxidize the formaldehyde glutathione adduct, S-hydroxymethylglutathione.GSNOR has since been identified in a variety of bacteria, yeasts,plants, and animals and is well conserved. The proteins from E. coli, S.cerevisiae and mouse macrophages share over 60% amino acid sequenceidentity. GSNOR activity (i.e., decomposition of GSNO when NADH ispresent as a required cofactor) has been detected in E. coli, in mousemacrophages, in mouse endothelial cells, in mouse smooth muscle cells,in yeasts, and in human HeLa, epithelial, and monocyte cells. HumanGSNOR nucleotide and amino acid sequence information can be obtainedfrom the National Center for Biotechnology Information (NCBI) databasesunder Accession Nos. M29872, NM_000671. Mouse GSNOR nucleotide and aminoacid sequence information can be obtained from NCBI databases underAccession Nos. NM_007410. In the nucleotide sequence, the start site andstop site are underlined. CDS designates coding sequence. SNP designatessingle nucleotide polymorphism. Other related GSNOR nucleotide and aminoacid sequences, including those of other species, can be found in U.S.Patent Application 2005/0014697.

In accord with the present invention, GSNOR has been shown to functionin vivo and in vitro to metabolize S-nitrosoglutathione (GSNO) andprotein S-nitrosothiols (SNOs) to modulate NO bioactivity, bycontrolling the intracellular levels of low mass NO donor compounds andpreventing protein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of this enzyme potentiatesbioactivity in diseases in which NO donor therapy is indicated, inhibitsthe proliferation of pathologically proliferating cells, and increasesNO bioactivity in diseases where this is beneficial.

The present invention provides pharmaceutical agents that are potentinhibitors of GSNOR. In particular, provided are substituted bicyclicaromatic analogs having the structure depicted below (Formula 1), or apharmaceutically acceptable salt, stereoisomer, prodrug, metabolite, orN-oxide thereof.

-   Wherein-   R₁ is selected from the group consisting of H, F, and Cl;-   R_(2a) and R_(2b) are independently selected from the group    consisting of H, F, Cl, Br, Me, OCH₃, and cyano;-   R_(2c) is selected from the group consisting of H, F, Cl, Br, Me,    and OCH₃;-   X is selected from the group consisting of

-   A is selected from the group consisting of

-   R₃ is selected from the group consisting of F, Cl, Br, CH₃, CF₃,    OCH₃, cyano, N(CH₃)₂, and morpholino;-   n is selected from the group consisting of 0, 1, and 2;-   R₄ is selected from the group consisting of H, F, Cl, Br, CH₃, CF₃,    OCH₃, cyano, N(CH₃)₂, and morpholino; and-   with the proviso that when X is

and A is COOH, then at least one of R₁, R_(2a), R_(2b), R_(2c), and R₄is not hydrogen, or n must be >0, and R₃ when meta to naphthalene,cannot be CH₃.

As used in this context, the term “analog” refers to a compound havingsimilar chemical structure and function as compounds of Formula I thatretains the substituted bicyclic aromatic ring system.

Some analogs of the invention can also exist in various isomeric forms,including configurational, geometric, and conformational isomers, aswell as existing in various tautomeric forms, particularly those thatdiffer in the point of attachment of a hydrogen atom. As used herein,the term “isomer” is intended to encompass all isomeric forms of acompound including tautomeric forms of the compound.

Illustrative compounds having asymmetric centers can exist in differentenantiomeric and diastereomeric forms. A compound can exist in the formof an optical isomer or a diastereomer. Accordingly, the inventionencompasses compounds in the forms of their optical isomers,diastereomers and mixtures thereof, including racemic mixtures.

It should be noted that if there is a discrepancy between a depictedstructure and a name given to that structure, the depicted structurecontrols. In addition, if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold, wedged,or dashed lines, the structure or portion of the structure is to beinterpreted as encompassing all stereoisomers of the described compound.

B. S-Nitrosoglutathione Reductase Inhibitors

1. Inventive Compounds

In one of its aspects the present invention provides a compound having astructure shown in Formula 1, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

-   Wherein-   R₁ is selected from the group consisting of H, F, and Cl;-   R_(2a) and R_(2b) are independently selected from the group    consisting of H, F, Cl, Br, Me, OCH₃, and cyano;-   R_(2c) is selected from the group consisting of H, F, Cl, Br, Me,    and OCH₃;-   X is selected from the group consisting of

-   A is selected from the group consisting of

-   R₃ is selected from the group consisting of F, Cl, Br, CH₃, CF₃,    OCH₃, cyano, N(CH₃)₂, and morpholino;-   n is selected from the group consisting of 0, 1, and 2;-   R₄ is selected from the group consisting of H, F, Cl, Br, CH₃, CF₃,    OCH₃, cyano, N(CH₃)₂, and morpholino; and    with the proviso that when X is

and A is COOH, then at least one of R₁, R_(2a), R_(2b), R_(2c), R₄ isnot hydrogen, or n must be >0, and R₃ when meta to naphthalene, cannotbe CH₃.

In a further aspect of the invention, X is selected from the groupconsisting of

In a further aspect of the invention, X is

In a further aspect of the invention, R_(2c) is hydrogen.

In a further aspect of the invention, R_(2b) is hydrogen.

In a further aspect of the invention, the compound of Formula 1 has thestructure shown in Formula 2:

In a further aspect of the invention, R₁ of Formula 2 is selected fromthe group consisting of H and F;

In a further aspect of the invention, R_(2a) of Formula 2 is selectedfrom the group consisting of H, F, Cl, Br, and cyano;

In a further aspect of the invention, R_(2b) of Formula 2 is selectedfrom the group consisting of H, F, and Cl;

In a further aspect of the invention, R_(2c) of Formula 2 is H;

In a further aspect of the invention, R₄ of Formula 2 is selected fromthe group consisting of H, F, Cl, and cyano.

In a further aspect of the invention, R₁ of Formula 2 is selected fromthe group consisting of F and Cl.

In a further aspect of the invention, R_(2a) of Formula 2 is selectedfrom the group consisting of F, Cl, Br, Me, OCH₃, and cyano.

In a further aspect of the invention, R_(2b) of Formula 2 is selectedfrom the group consisting of F, Cl, Br, Me, OCH₃, and cyano.

In a further aspect of the invention, R_(2c) of Formula 2 is selectedfrom the group consisting of F, Cl, Br, Me, and OCH₃;

In a further aspect of the invention, R₄ of Formula 2 is selected fromthe group consisting of F, Cl, Br, CH₃, CF₃, OCH₃, cyano, N(CH₃)₂, andmorpholino;

In a further aspect of the invention, the compound of Formula 1 has thestructure shown in Formula 3

In a further aspect of the invention, X of Formula 3 is

In a further aspect of the invention, A of Formula 3 is COOH.

In a further aspect of the invention, the compound of Formula 3 has thestructure shown in Formula 4:

In a further aspect of the invention, R_(2c) of Formula 4 is H.

In a further aspect of the invention, R_(2b) of Formula 4 is H.

In a further aspect of the invention, R₁ of Formula 4 is selected fromthe group consisting of H and F.

In a further aspect of the invention, R_(2b) of Formula 4 is selectedfrom the group consisting of H, F, and Cl.

In a further aspect of the invention, R_(2c) of Formula 4 is H;

In a further aspect of the invention, R₄ of Formula 4 is selected fromthe group consisting of H, F, Cl, and cyano.

In a further aspect of the invention, the compound of Formula 1 has thestructure shown in Formula 5

In a further aspect of the invention, X of Formula 5 is

In a further aspect of the invention, A of Formula 5 is COOH.

In a further aspect of the invention, the compound of Formula 5 has thestructure shown in Formula 6:

In a further aspect of the invention, R_(2c) of Formula 6 is H.

In a further aspect of the invention, R_(2b) of Formula 6 is H.

In a further aspect of the invention, R_(2a) of Formula 6 is selectedfrom the group consisting of H, F, Cl, Br, and cyano.

In a further aspect of the invention, R_(2b) of Formula 6 is selectedfrom the group consisting of H, F, and Cl.

In a further aspect of the invention, R_(2c) of Formula 6 is H;

In a further aspect of the invention, R₄ of Formula 6 is selected fromthe group consisting of H, F, Cl, and cyano.

In one of its aspects the present invention provides a compound having astructure shown in Formula 7, or a pharmaceutically acceptable salt,stereoisomer, prodrug, metabolite, or N-oxide thereof:

-   wherein-   Z₁ is selected from the group consisting of CR_(2a) and N;-   Z₂ is selected from the group consisting of CR_(2b) and N;-   Z₃ is selected from the group consisting of CR_(2c) and N;-   with the proviso that at least 1 of Z₁, Z₂, or Z₃ must be N;-   m is selected from the group consisting of 0, 1, 2, or 3;-   R₁ is independently selected from the group consisting of chloro,    fluoro, and bromo;-   R_(2a), R_(2b), and R_(2c), are independently selected from the    group consisting of hydrogen, halogen, C₁-C₃ alkyl, fluorinated    C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and N(CH₃)₂;-   X is selected from the group consisting of

-   n is selected from 0, 1, and 2;-   R₃ is independently selected from the group consisting of halogen,    C₁-C₃ alkyl, fluorinated C₁-C₃ alkyl, cyano, C₁-C₃ alkoxy, and    NR₄R_(4′) where R₄ and R_(4′) are independently selected from the    group consisting of C₁-C₃ alkyl, or R₄ when taken together with    R_(4′) form a ring with 3 to 6 members;-   A is selected from the group consisting of

One aspect of the invention includes compounds of Formula 7 wherein Z₁,Z₂, and Z₃ are all CR_(2a-c).

One aspect of the invention includes compounds of Formula 7 wherein Z₁,Z₂, and Z₃ are all CR_(2a-c) and wherein X has an expanded definition toinclude nitrogen containing 6-membered aromatic rings.

In a further aspect of the invention, compounds of Formula 1 include butare not limited to:

-   3-chloro-4-(6-hydroxynaphthalen-2-yl)benzoic acid;-   3-fluoro-4-(6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(6-hydroxynaphthalen-2-yl)-3-methoxybenzoic acid;-   3-(dimethylamino)-4-(6-hydroxynaphthalen-2-yl)benzoic acid;-   3-cyano-4-(6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(6-hydroxynaphthalen-2-yl)-3-morpholinobenzoic acid;-   4-(1-bromo-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(6-hydroxy-1-methylnaphthalen-2-yl)benzoic acid;-   4-(1-cyano-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(6-hydroxy-3-methoxynaphthalen-2-yl)benzoic acid;-   4-(1-chloro-6-hydroxynaphthalen-2-yl)benzoic acid;-   6-(4-(1H-tetrazol-5-yl)phenyl)naphthalen-2-ol;-   5-(6-hydroxynaphthalen-2-yl)picolinic acid;-   6-(6-hydroxynaphthalen-2-yl)nicotinic acid;-   5-(6-hydroxynaphthalen-2-yl)pyrazine-2-carboxylic acid;-   2-(6-hydroxynaphthalen-2-yl)pyrimidine-5-carboxylic acid;-   6-(6-hydroxynaphthalen-2-yl)pyridazine-3-carboxylic acid;-   5-(6-hydroxynaphthalen-2-yl)pyrimidine-2-carboxylic acid;-   6-(1H-benzo[d][1,2,3]triazol-6-yl)naphthalen-2-ol;-   4-(6-hydroxynaphthalen-2-yl)-3-(trifluoromethyl)benzoic acid;-   3-chloro-4-(3-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(3-chloro-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(3-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(6-hydroxy-1-methoxynaphthalen-2-yl)benzoic acid;-   4-(1-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(6-hydroxy-3-methylnaphthalen-2-yl)benzoic acid;-   4-(1-cyano-5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(1-cyano-6-hydroxynaphthalen-2-yl)-3-fluorobenzoic acid;-   3-chloro-4-(5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid;-   4-(5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid;-   3-fluoro-4-(5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid; and-   4-(5-fluoro-6-hydroxynaphthalen-2-yl)-3-methylbenzoic acid.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchsubstituent. Combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The compounds described herein may have asymmetric centers. Compounds ofthe present invention containing an asymmetrically substituted atom maybe isolated in optically active or racemic forms. It is well known inthe art how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.Many geometric isomers of olefins, C═N double bonds, and the like canalso be present in the compounds described herein, and all such stableisomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral, diastereomeric, racemic, and geometricisomeric forms of a structure are intended, unless the specificstereochemistry or isomeric form is specifically indicated. Alltautomers of shown or described compounds are also considered to be partof the present invention.

It is to be understood that isomers arising from such asymmetry (e.g.,all enantiomers and diastereomers) are included within the scope of theinvention, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Furthermore, the structures andother compounds and moieties discussed in this application also includeall tautomers thereof. Alkenes can include either the E- or Z-geometry,where appropriate.

2. Representative Compounds

Examples 1-32 list representative substituted bicyclic aromaticcompounds analogs of Formula I. The synthetic methods that can be usedto prepare each compound are detailed in Examples 1-32. Supporting massspectrometry data and/or proton NMR data for each compound is alsoincluded in Examples 1-32. GSNOR inhibitor activity was determined bythe assay described in Example 34 and IC₅₀ values were obtained. GSNORinhibitor compounds in Examples 1-33 had an IC₅₀ of about <5 μM. GSNORinhibitor compounds in Examples 1-3, 5, 6, 12, 15, 17, 18, 20-33 had anIC₅₀ of about <0.1 μM. GSNOR inhibitor compounds in Examples 1, 2, 6,12, 15, 17, 21-23, 25, 27-32 had an IC₅₀ of about <0.05 μM.

C. Definitions

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

The term “acyl” includes compounds and moieties that contain the acetylradical (CH₃CO—) or a carbonyl group to which a straight or branchedchain lower alkyl residue is attached.

The term “alkyl” as used herein refers to a straight or branched chain,saturated hydrocarbon having the indicated number of carbon atoms. Forexample, (C₁-C₆) alkyl is meant to include, but is not limited tomethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tent-butyl, pentyl,isopentyl, neopentyl, hexyl, isohexyl, and neohexyl. An alkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein.

The term “alkenyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one double bond. Examples of a (C₂-C₈) alkenyl group include,but are not limited to, ethylene, propylene, 1-butylene, 2-butylene,isobutylene, sec-butylene, 1-pentene, 2-pentene, isopentene, 1-hexene,2-hexene, 3-hexene, isohexene, 1-heptene, 2-heptene, 3-heptene,isoheptene, 1-octene, 2-octene, 3-octene, 4-octene, and isooctene. Analkenyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkynyl” as used herein refers to a straight or branched chainunsaturated hydrocarbon having the indicated number of carbon atoms andat least one triple bond. Examples of a (C₂-C₈) alkynyl group include,but are not limited to, acetylene, propyne, 1-butyne, 2-butyne,1-pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne,2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne, and 4-octyne. Analkynyl group can be unsubstituted or optionally substituted with one ormore substituents as described herein.

The term “alkoxy” as used herein refers to an —O-alkyl group having theindicated number of carbon atoms. For example, a (C₁-C₆) alkoxy groupincludes —O-methyl, —O-ethyl, —O-propyl, —O-isopropyl, —O-butyl,—O-sec-butyl, —O-tert-butyl, —O-pentyl, —O-isopentyl, —O-neopentyl,—O-hexyl, —O-isohexyl, and —O-neohexyl.

The term “aminoalkyl” as used herein, refers to an alkyl group(typically one to six carbon atoms) wherein one or more of the C₁-C₆alkyl group's hydrogen atoms is replaced with an amine of formula—N(R^(c))₂, wherein each occurrence of R^(c) is independently —H or(C₁-C₆) alkyl. Examples of aminoalkyl groups include, but are notlimited to, —CH₂NH₂, —CH₂CH₂NH₂, —CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂NH₂,—CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂CH₂CH₂CH₂NH₂, —CH₂CH₂CH₂N(CH₃)₂,t-butylaminomethyl, isopropylaminomethyl, and the like.

The term “aryl” as used herein refers to a 5- to 14-membered monocyclic,bicyclic, or tricyclic aromatic ring system. Examples of an aryl groupinclude phenyl and naphthyl. An aryl group can be unsubstituted oroptionally substituted with one or more substituents as described hereinbelow. Examples of aryl groups include phenyl or aryl heterocycles suchas, pyrrole, furan, thiophene, thiazole, isothiazole, imidazole,triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

As used herein, the term “bioactivity” indicates an effect on one ormore cellular or extracellular process (e.g., via binding, signaling,etc.) which can impact physiological or pathophysiological processes.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxy” or “carboxyl” means a —COOH group or carboxylic acid.

“Acidic moiety” as used herein is defined as a carboxylic acid or acarboxylic acid bioisostere. Bioisosteres are substituents or groupswith similar physical or chemical properties which produce broadlysimilar biological properties to a chemical compound. For a review ofbioisosteres, see J. Med. Chem, 2011, 54, 2529-2591. Examples of “acidicmoiety” include but are not limited to

“Pharmacophore” is defined as “a set of structural features in amolecule that is recognized at a receptor site and is responsible forthat molecule's biological activity” (Gund, Prog. Mol. Subcell. Biol.,5: pp 117-143 (1977)).

The term “C_(m)-C_(n)” means “m” number of carbon atoms to “n” number ofcarbon atoms. For example, the term “C₁-C₆” means one to six carbonatoms (C₁, C₂, C₃, C₄, C₅, or C₆). The term “C₂-C₆” includes two to sixcarbon atoms (C₂, C₃, C₄, C₅, or C₆). The term “C₃-C₆” includes three tosix carbon atoms (C₃, C₄, C₅, or C₆).

The term “cycloalkyl” as used herein refers to a 3- to 14-memberedsaturated or unsaturated non-aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system. Included in this class are cycloalkyl groupswhich are fused to a benzene ring. Representative cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl,cyclopentyl, cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,1,3-cyclohexadienyl, cycloheptyl, cycloheptenyl, 1,3-cycloheptadienyl,1,4-cycloheptadienyl, -1,3,5-cycloheptatrienyl, cyclooctyl,cyclooctenyl, 1,3-cyclooctadienyl, 1,4-cyclooctadienyl,-1,3,5-cyclooctatrienyl, decahydronaphthalene, octahydronaphthalene,hexahydronaphthalene, octahydroindene, hexahydroindene, tetrahydroinden,decahydrobenzocycloheptene, octahydrobenzocycloheptene,hexahydrobenzocycloheptene, tetrahydrobenzocyclopheptene,dodecahydroheptalene, decahydroheptalene, octahydroheptalene,hexahydroheptalene, tetrahydroheptalene, (1s,3s)-bicyclo[1.1.0]butane,bicyclo[1.1.1]pentane, bicyclo[2.1.1]hexane, Bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.1.1]heptane, bicyclo[3.2.1]octane,bicyclo[3.3.1]nonane, bicyclo[3.3.2]decane, bicyclo [3.3.]undecane,bicyclo[4.2.2]decane, and bicyclo[4.3.1]decane. A cycloalkyl group canbe unsubstituted or optionally substituted with one or more substituentsas described herein below.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.

The term “haloalkyl,” as used herein, refers to a C₁-C₆ alkyl groupwherein from one or more of the C₁-C₆ alkyl group's hydrogen atom isreplaced with a halogen atom, which can be the same or different.Examples of haloalkyl groups include, but are not limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl,pentachloroethyl, and 1,1,1-trifluoro-2-bromo-2-chloroethyl.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chainalkyl, or combinations thereof, consisting of carbon atoms and from oneto three heteroatoms selected from the group consisting of O, N, and S,and wherein the nitrogen and sulfur atoms may optionally be oxidized andthe nitrogen heteroatom may optionally be quaternized. The heteroatom(s)O, N, and S can be placed at any position of the heteroalkyl group.Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, and—CH₂—CH═N—OCH₃. Up to two heteroatoms can be consecutive, for example,—CH₂—NH—OCH₃. When a prefix such as (C₂-C₈) is used to refer to aheteroalkyl group, the number of carbons (2 to 8, in this example) ismeant to include the heteroatoms as well. For example, a C₂-heteroalkylgroup is meant to include, for example, —CH₂OH (one carbon atom and oneheteroatom replacing a carbon atom) and —CH₂SH.

To further illustrate the definition of a heteroalkyl group, where theheteroatom is oxygen, a heteroalkyl group can be an oxyalkyl group. Forinstance, (C₂₋C₅) oxyalkyl is meant to include, for example —CH₂—O—CH₃(a C₃-oxyalkyl group with two carbon atoms and one oxygen replacing acarbon atom), —CH₂CH₂CH₂CH₂OH, —OCH₂CH₂OCH₂CH₂OH, —OCH₂CH(OH)CH₂OH, andthe like.

The term “heteroaryl” as used herein refers to an aromatic heterocyclering of 5 to 14 members and having at least one heteroatom selected fromnitrogen, oxygen, and sulfur, and containing at least 1 carbon atom,including monocyclic, bicyclic, and tricyclic ring systems.Representative heteroaryls are triazolyl, tetrazolyl, oxadiazolyl,pyridyl, furyl, benzofuranyl, thienyl, benzothienyl, quinolinyl,pyrrolyl, indolyl, oxazolyl, benzoxazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isoxazolyl, pyrazolyl, isothiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl,phthalazinyl, quinazolinyl, pyrimidyl, azepinyl, oxepinyl, quinoxalinyland oxazolyl. A heteroaryl group can be unsubstituted or optionallysubstituted with one or more substituents as described herein below.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), and sulfur (S).

As used herein, the term “heterocycle” refers to 3- to 14-membered ringsystems which are either saturated, unsaturated, or aromatic, and whichcontains from 1 to 4 heteroatoms independently selected from nitrogen,oxygen, and sulfur, and wherein the nitrogen and sulfur heteroatoms canbe optionally oxidized, and the nitrogen heteroatom can be optionallyquaternized, including monocyclic, bicyclic, and tricyclic ring systems.The bicyclic and tricyclic ring systems may encompass a heterocycle orheteroaryl fused to a benzene ring. The heterocycle can be attached viaany heteroatom or carbon atom, where chemically acceptable. Heterocyclesinclude heteroaryls as defined above. Representative examples ofheterocycles include, but are not limited to, aziridinyl, oxiranyl,thiiranyl, triazolyl, tetrazolyl, azirinyl, diaziridinyl, diazirinyl,oxaziridinyl, azetidinyl, azetidinonyl, oxetanyl, thietanyl,piperidinyl, piperazinyl, morpholinyl, pyrrolyl, oxazinyl, thiazinyl,diazinyl, dioxanyl, triazinyl, tetrazinyl, imidazolyl, tetrazolyl,pyrrolidinyl, isoxazolyl, furanyl, furazanyl, pyridinyl, oxazolyl,benzoxazolyl, benzisoxazolyl, thiazolyl, benzthiazolyl, thienyl,pyrazolyl, triazolyl, pyrimidinyl, benzimidazolyl, isoindolyl,indazolyl, benzodiazolyl, benzotriazolyl, benzoxazolyl, benzisoxazolyl,purinyl, indolyl, isoquinolinyl, quinolinyl, and quinazolinyl. Aheterocycle group can be unsubstituted or optionally substituted withone or more substituents as described herein below.

The term “heterocycloalkyl,” by itself or in combination with otherterms, represents, unless otherwise stated, cyclic versions of“heteroalkyl.” Additionally, a heteroatom can occupy the position atwhich the heterocycle is attached to the remainder of the molecule.Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like.

The term “hydroxyalkyl,” as used herein, refers to an alkyl group havingthe indicated number of carbon atoms wherein one or more of the hydrogenatoms in the alkyl group is replaced with an —OH group. Examples ofhydroxyalkyl groups include, but are not limited to, —CH₂OH, —CH₂CH₂OH,—CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂OH, —CH₂CH₂CH₂CH₂CH₂OH,—CH₂CH₂CH₂CH₂CH₂CH₂OH, and branched versions thereof.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, N-oxide, or amine oxide, refers to a compound derivedfrom a tertiary amine by the attachment of one oxygen atom to thenitrogen atom, R₃N⁺—O⁻. By extension the term includes the analogousderivatives of primary and secondary amines.

As used herein and unless otherwise indicated, the term “stereoisomer”means one stereoisomer of a compound that is substantially free of otherstereoisomers of that compound. For example, a stereomerically purecompound having one chiral center will be substantially free of theopposite enantiomer of the compound. A stereomerically pure compoundhaving two chiral centers will be substantially free of otherdiastereomers of the compound. In some embodiments, a stereomericallypure compound comprises greater than about 80% by weight of onestereoisomer of the compound and less than about 20% by weight of otherstereoisomers of the compound, for example greater than about 90% byweight of one stereoisomer of the compound and less than about 10% byweight of the other stereoisomers of the compound, or greater than about95% by weight of one stereoisomer of the compound and less than about 5%by weight of the other stereoisomers of the compound, or greater thanabout 97% by weight of one stereoisomer of the compound and less thanabout 3% by weight of the other stereoisomers of the compound.

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment”. A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

As used herein, “modulate” is meant to refer to an increase or decreasein the levels of a peptide or a polypeptide, or to increase or decreasethe stability or activity of a peptide or a polypeptide. The term“inhibit” is meant to refer to a decrease in the levels of a peptide ora polypeptide or to a decrease in the stability or activity of a peptideor a polypeptide. In preferred embodiments, the peptide which ismodulated or inhibited is S-nitrosoglutathione (GSNO) or proteinS-nitrosothiols (SNOs).

As used here, the terms “nitric oxide” and “NO” encompass unchargednitric oxide and charged nitric oxide species, particularly includingnitrosonium ion (NO⁺) and nitroxyl ion (NO⁻). The reactive form ofnitric oxide can be provided by gaseous nitric oxide. Compounds havingthe structure X—NO_(y) wherein X is a nitric oxide releasing,delivering, or transferring moiety, including any and all such compoundswhich provide nitric oxide to its intended site of action in a formactive for their intended purpose, and Y is 1 or 2.

Repair” means recovering of structural integrity and normal physiologicfunction. By way of example, the oral and upper airway respiratoryepithelium can repair damage done by thermal injury or viral infection.

“Regeneration” means the ability of an organ to enter non-malignantcellular, vascular and stromal growth to restore functional organtissue. By way of example, wound healing involves regeneration of tissueand organs (e.g. skin, gastric and intestinal mucosa), as does bonefollowing fracture, and the liver following partial surgical removal andexposure to infectious or toxic insult.

As utilized herein, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of a federal or a state government orlisted in the U.S. Pharmacopoeia or other generally recognizedpharmacopoeia for use in animals and, more particularly, in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered and includes, but is not limitedto such sterile liquids as water and oils.

A “pharmaceutically acceptable salt” or “salt” of a compound of theinvention is a product of the disclosed compound that contains an ionicbond, and is typically produced by reacting the disclosed compound witheither an acid or a base, suitable for administering to a subject. Apharmaceutically acceptable salt can include, but is not limited to,acid addition salts including hydrochlorides, hydrobromides, phosphates,sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates,arylalkylsulfonates, acetates, benzoates, citrates, maleates, fumarates,succinates, lactates, and tartrates; alkali metal cations such as Li,Na, and K, alkali earth metal salts such as Mg or Ca, or organic aminesalts.

A “pharmaceutical composition” is a formulation comprising the disclosedcompounds in a form suitable for administration to a subject. Apharmaceutical composition of the invention is preferably formulated tobe compatible with its intended route of administration. Examples ofroutes of administration include, but are not limited to, oral andparenteral, e.g., intravenous, intradermal, subcutaneous, inhalation,topical, transdermal, transmucosal, and rectal administration.

The term “substituted, ” as used herein, means that any one or morehydrogens on the designated atom is replaced with a selection from theindicated group, provided that the designated atom's normal valency isnot exceeded, and that the substitution results in a stable compound.When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom arereplaced. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

Substituents for the groups referred to as alkyl, heteroalkyl, alkylene,alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl can be selected from a variety of groups including—OR^(d)′, ═O, ═NR^(d)′, ═N—OR^(d)′, —NR^(d)′R^(d)″, —SR^(d)′, -halo,-SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′, —C(O)R^(d)′, —CO₂R^(d)′,—CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)′″C(O)NR^(d)′R^(d)″, —NR^(d)′″SO₂NR^(d)′R^(d)″,—NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH, —NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′,—S(O)R^(d)′, —SO₂R^(d)′, —SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and—NO₂, in a number ranging from zero to three, with those groups havingzero, one or two substituents being exemplary.

R^(d)′, R^(d)″, and R^(d)′″ each independently refer to hydrogen,unsubstituted (C₁-C₈)alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, and aryl substituted with one to three substituentsselected from -halo, unsubstituted alkyl, unsubstituted alkoxy,unsubstituted thioalkoxy, and unsubstituted aryl (C₁-C₄)alkyl. WhenR^(d)′ and R^(d)″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR^(d)′R^(d)″ can represent 1-pyrrolidinyl or4-morpholinyl.

Typically, an alkyl or heteroalkyl group will have from zero to threesubstituents, with those groups having two or fewer substituents beingexemplary of the present invention. An alkyl or heteroalkyl radical canbe unsubstituted or monosubstituted. In some embodiments, an alkyl orheteroalkyl radical will be unsubstituted.

Exemplary substituents for the alkyl and heteroalkyl radicals include,but are not limited to —OR^(d)′, ═O, ═NR^(d)′, ═N—OR^(d)′,—NR^(d)′R^(d)″, —SR^(d)′, -halo, —SiR^(d)′R^(d)″R^(d)′″, —OC(O)R^(d)′,—C(O)R^(d)′, —CO₂R^(d)′, —CONR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″,—NR^(d)″C(O)R^(d)′, —NR^(d)′″C(O)NR^(d)′R^(d)″,—NR^(d)′″SO₂NR^(d)′R^(d)″, —NR^(d)″CO₂R^(d)′, —NHC(NH₂)═NH,—NR^(a)′C(NH₂)═NH, —NHC(NH₂)═NR^(d)′, —S(O)R^(d)′, —SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and —NO₂, where R^(d)′,R^(d)″, and R^(d)′″ are as defined above. Typical substituents can beselected from: —OR^(d)′, ═O, —NR^(d)′R^(d)″, -halo, —OC(O)R^(d)′,—CO₂R^(d)′, —C(O)NR^(d)′R^(d)″, —OC(O)NR^(d)′R^(d)″, —NR^(d)″C(O)R^(d)′,—NR^(d)″CO₂R^(d)′, —NR^(d)′″SO₂NR^(d)′R^(d)″, —SO₂R^(d)′,—SO₂NR^(d)′R^(d)″, —NR^(d)″SO₂R^(d)′, —CN, and —NO₂.

Similarly, substituents for the aryl and heteroaryl groups are variedand selected from: -halo, —OR^(e)′, —OC(O)R^(e)′, —NR^(e)′R^(e)″,—SR^(e)′, —R^(e)′, —CN, —NO₂, —CO₂R^(e)′, —C(O)NR^(e)′R^(e)″,—C(O)R^(e)′, —OC(O)NR^(e)′R^(e)″, —NR^(e)″C(O)R^(e)′, —NR^(e)″CO₂R^(e)′,—NR^(e)′″C(O)NR^(e)′R^(e)″, —NR^(e)′″SO₂NR^(e)′R^(e)″, —NHC(NH₂)═NH,—NR^(e)′C(NH₂)═NH, —NH—C(NH₂)═NR^(e)′, —S(O)R^(e)′, —SO₂R^(e)′,—SO₂NR^(e)′R^(e)″, —NR^(e)″SO₂R^(e)′, —N₃, —CH(Ph)₂, perfluoroalkoxy,and perfluoro(C₁-C₄)alkyl, in a number ranging from zero to the totalnumber of open valences on the aromatic ring system.

R^(e)′, R^(e)″ and R^(e)′″ are independently selected from hydrogen,unsubstituted (C₁-C₈) alkyl, unsubstituted hetero(C₁-C₈) alkyl,unsubstituted aryl, unsubstituted heteroaryl, unsubstituted aryl(C₁-C₄)alkyl, and unsubstituted aryloxy(C₁-C₄) alkyl. Typically, an aryl orheteroaryl group will have from zero to three substituents, with thosegroups having two or fewer substituents being exemplary in the presentinvention. In one embodiment of the invention, an aryl or heteroarylgroup will be unsubstituted or monosubstituted. In another embodiment,an aryl or heteroaryl group will be unsubstituted.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringin an aryl or heteroaryl group as described herein may optionally bereplaced with a substituent of the formula -T-C(O)—(CH₂)_(q)—U—, whereinT and U are independently —NH—, —O—, —CH₂— or a single bond, and q is aninteger of from 0 to 2. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -J-(CH₂)_(r)—K—, wherein J and K areindependently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR^(f)′—, ora single bond, and r is an integer of from 1 to 3. One of the singlebonds of the new ring so formed may optionally be replaced with a doublebond. Alternatively, two of the substituents on adjacent atoms of thearyl or heteroaryl ring may optionally be replaced with a substituent ofthe formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR^(f)′—, —S—, —S(O)—, —S(O)₂—,or —S(O)₂NR^(a)′—. The substituent R^(f)′ in —NR^(f)′— and—S(O)₂NR^(f)′— is selected from hydrogen or unsubstituted (C₁-C₆) alkyl.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein the term “therapeutically effective amount” generallymeans the amount necessary to ameliorate at least one symptom of adisorder to be prevented, reduced, or treated as described herein. Thephrase “therapeutically effective amount” as it relates to the GSNORinhibitors of the present invention shall mean the GSNOR inhibitordosage that provides the specific pharmacological response for which theGSNOR inhibitor is administered in a significant number of subjects inneed of such treatment. It is emphasized that a therapeuticallyeffective amount of a GSNOR inhibitor that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

The term “biological sample” includes, but is not limited to, samples ofblood (e.g., serum, plasma, or whole blood), urine, saliva, sweat,breast milk, vaginal secretions, semen, hair follicles, skin, teeth,bones, nails, or other secretions, body fluids, tissues, or cells. Inaccordance with the invention, the levels of the GSNOR in the biologicalsample can be determined by the methods described in U.S. PatentApplication Publication No. 2005/0014697.

D. Pharmaceutical Compositions

The invention encompasses pharmaceutical compositions comprising atleast one compound of the invention described herein and at least onepharmaceutically acceptable carrier. Suitable carriers are described in“Remington: The Science and Practice, Twentieth Edition,” published byLippincott Williams & Wilkins, which is incorporated herein byreference. Pharmaceutical compositions according to the invention mayalso comprise one or more non-inventive compound active agents.

The pharmaceutical compositions of the invention can comprise novelcompounds described herein, the pharmaceutical compositions can compriseknown compounds which previously were not known to have GSNOR inhibitoractivity, or a combination thereof.

The compounds of the invention can be utilized in any pharmaceuticallyacceptable dosage form, including, but not limited to injectable dosageforms, liquid dispersions, gels, aerosols, ointments, creams,lyophilized formulations, dry powders, tablets, capsules, controlledrelease formulations, fast melt formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, mixed immediate release and controlled releaseformulations, etc. Specifically, the compounds of the inventiondescribed herein can be formulated: (a) for administration selected fromthe group consisting of oral, pulmonary, intravenous, intra-arterial,intrathecal, intra-articular, rectal, ophthalmic, colonic, parenteral,otic, intracisternal, intravaginal, intraperitoneal, local, buccal,nasal, and topical administration; (b) into a dosage form selected fromthe group consisting of liquid dispersions, gels, aerosols, ointments,creams, tablets, sachets, and capsules; (c) into a dosage form selectedfrom the group consisting of lyophilized formulations, dry powders, fastmelt formulations, controlled release formulations, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, and mixed immediate release and controlled releaseformulations; or (d) any combination thereof.

For respiratory infections, an inhalation formulation can be used toachieve high local concentrations. Formulations suitable for inhalationinclude dry power or aerosolized or vaporized solutions, dispersions, orsuspensions capable of being dispensed by an inhaler or nebulizer intothe endobronchial or nasal cavity of infected patients to treat upperand lower respiratory bacterial infections.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can comprise one or more of the followingcomponents: (1) a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,or other synthetic solvents; (2) antibacterial agents such as benzylalcohol or methyl parabens; (3) antioxidants such as ascorbic acid orsodium bisulfite; (4) chelating agents such asethylenediaminetetraacetic acid; (5) buffers such as acetates, citrates,or phosphates; and (5) agents for the adjustment of tonicity such assodium chloride or dextrose. The pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. A parenteral preparationcan be enclosed in ampoules, disposable syringes, or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use may comprisesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The pharmaceutical composition should bestable under the conditions of manufacture and storage and should bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The carrier can be a solvent or dispersion medium comprising, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion, and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol or sorbitol, and inorganic saltssuch as sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activereagent in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating at least one compound of the invention into a sterilevehicle that contains a basic dispersion medium and any other requiredingredients. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, both of which yield a powder of acompound of the invention plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed, for example, in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the compound of the invention can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser thatcontains a suitable propellant, e.g., a gas such as carbon dioxide, anebulized liquid, or a dry powder from a suitable device. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active reagents are formulated into ointments, salves, gels, orcreams as generally known in the art. The reagents can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In one embodiment, the compounds of the invention are prepared withcarriers that will protect against rapid elimination from the body. Forexample, a controlled release formulation can be used, includingimplants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

Additionally, suspensions of the compounds of the invention may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils, such as sesame oil, orsynthetic fatty acid esters, such as ethyl oleate, triglycerides, orliposomes. Non-lipid polycationic amino polymers may also be used fordelivery. Optionally, the suspension may also include suitablestabilizers or agents to increase the solubility of the compounds andallow for the preparation of highly concentrated solutions.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of thecompound of the invention calculated to produce the desired therapeuticeffect in association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on the unique characteristics of the compound ofthe invention and the particular therapeutic effect to be achieved, andthe limitations inherent in the art of compounding such an active agentfor the treatment of individuals.

Pharmaceutical compositions according to the invention comprising atleast one compound of the invention can comprise one or morepharmaceutical excipients. Examples of such excipients include, but arenot limited to binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art. Exemplary excipientsinclude: (1) binding agents which include various celluloses andcross-linked polyvinylpyrrolidone, microcrystalline cellulose, such asAvicel® PH101 and Avicel® PH102, silicified microcrystalline cellulose(ProSolv SMCC™), gum tragacanth and gelatin; (2) filling agents such asvarious starches, lactose, lactose monohydrate, and lactose anhydrous;(3) disintegrating agents such as alginic acid, Primogel, corn starch,lightly crosslinked polyvinyl pyrrolidone, potato starch, maize starch,and modified starches, croscarmellose sodium, cross-povidone, sodiumstarch glycolate, and mixtures thereof; (4) lubricants, including agentsthat act on the flowability of a powder to be compressed, includemagnesium stearate, colloidal silicon dioxide, such as Aerosil® 200,talc, stearic acid, calcium stearate, and silica gel; (5) glidants suchas colloidal silicon dioxide; (6) preservatives, such as potassiumsorbate, methylparaben, propylparaben, benzoic acid and its salts, otheresters of parahydroxybenzoic acid such as butylparaben, alcohols such asethyl or benzyl alcohol, phenolic compounds such as phenol, orquaternary compounds such as benzalkonium chloride; (7) diluents such aspharmaceutically acceptable inert fillers, such as microcrystallinecellulose, lactose, dibasic calcium phosphate, saccharides, and/ormixtures of any of the foregoing; examples of diluents includemicrocrystalline cellulose, such as Avicel® PH101 and Avicel® PH102;lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose®DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch;sorbitol; sucrose; and glucose; (8) sweetening agents, including anynatural or artificial sweetener, such as sucrose, saccharin sucrose,xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame; (9)flavoring agents, such as peppermint, methyl salicylate, orangeflavoring, Magnasweet® (trademark of MAFCO), bubble gum flavor, fruitflavors, and the like; and (10) effervescent agents, includingeffervescent couples such as an organic acid and a carbonate orbicarbonate. Suitable organic acids include, for example, citric,tartaric, malic, fumaric, adipic, succinic, and alginic acids andanhydrides and acid salts. Suitable carbonates and bicarbonates include,for example, sodium carbonate, sodium bicarbonate, potassium carbonate,potassium bicarbonate, magnesium carbonate, sodium glycine carbonate,L-lysine carbonate, and arginine carbonate. Alternatively, only thesodium bicarbonate component of the effervescent couple may be present.

E. Kits Comprising the Compositions of the Invention

The present invention also encompasses kits comprising the compositionsof the invention. Such kits can comprise, for example, (1) at least onecompound of the invention; and (2) at least one pharmaceuticallyacceptable carrier, such as a solvent or solution. Additional kitcomponents can optionally include, for example: (1) any of thepharmaceutically acceptable excipients identified herein, such asstabilizers, buffers, etc., (2) at least one container, vial, or similarapparatus for holding and/or mixing the kit components; and (3) deliveryapparatus, such as an inhaler, nebulizer, syringe, etc.

F. Methods of Preparing Compounds of the Invention

The compounds of the invention can readily be synthesized using knownsynthetic methodologies or via a modification of known syntheticmethodologies. As would be readily recognized by a skilled artisan, themethodologies described below allow the synthesis of substitutedbicyclic aromatic compoundss having a variety of substituents. Exemplarysynthetic methods are described in the Examples section below.

If needed, further purification and separation of enantiomers anddiastereomers can be achieved by routine procedures known in the art.Thus, for example, the separation of enantiomers of a compound can beachieved by the use of chiral HPLC and related chromatographictechniques. Diastereomers can be similarly separated. In some instances,however, diastereomers can simply be separated physically, such as, forexample, by controlled precipitation or crystallization.

The process of the invention, when carried out as prescribed herein, canbe conveniently performed at temperatures that are routinely accessiblein the art. In one embodiment, the process is performed at a temperaturein the range of about 25° C. to about 110° C. In another embodiment, thetemperature is in the range of about 40° C. to about 100° C. In yetanother embodiment, the temperature is in the range of about 50° C. toabout 95° C.

Synthetic steps that require a base are carried out using any convenientorganic or inorganic base. Typically, the base is not nucleophilic.Thus, in one embodiment, the base is selected from carbonates,phosphates, hydroxides, alkoxides, salts of disilazanes, and tertiaryamines.

The process of the invention, when performed as described herein, can besubstantially complete after several minutes to after several hoursdepending upon the nature and quantity of reactants and reactiontemperature. The determination of when the reaction is substantiallycomplete can be conveniently evaluated by ordinary techniques known inthe art such as, for example, HPLC, LCMS, TLC, and ¹H NMR.

G. Methods of Treatment

The invention encompasses methods of preventing or treating (e.g.,alleviating one or more symptoms of) medical conditions through use ofone or more of the disclosed compounds. The methods compriseadministering a therapeutically effective amount of a compound of theinvention to a patient in need. The compositions of the invention canalso be used for prophylactic therapy.

The compound of the invention used in the methods of treatment accordingto the invention can be: (1) a novel compound described herein, or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof; (2) acompound which was known prior to the present invention, but wherein itwas not known that the compound is a GSNOR inhibitor, or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof; or (3) acompound which was known prior to the present invention, and wherein itwas known that the compound is a GSNOR inhibitor, but wherein it was notknown that the compound is useful for the methods of treatment describedherein, or a pharmaceutically acceptable salt, a stereoisomer, aprodrug, a metabolite, or an N-oxide thereof.

The patient can be any animal, domestic, livestock, or wild, including,but not limited to cats, dogs, horses, pigs, and cattle, and preferablyhuman patients. As used herein, the terms patient and subject may beused interchangeably.

As used herein, “treating” describes the management and care of apatient for the purpose of combating a disease, condition, or disorderand includes the administration of a compound of the present inventionto prevent the onset of the symptoms or complications, alleviating thesymptoms or complications, or eliminating the disease, condition, ordisorder. More specifically, “treating” includes reversing, attenuating,alleviating, minimizing, suppressing, or halting at least onedeleterious symptom or effect of a disease (disorder) state, diseaseprogression, disease causative agent (e.g., bacteria or viruses), orother abnormal condition. Treatment is continued as long as symptomsand/or pathology ameliorate.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg/kg to 10 g/kg and often ranges from 10 μg/kg to 1 g/kgor 10 μg/kg to 100 mg/kg body weight of the subject being treated, perday.

H. GSNOR Uses

In subjects with deleteriously high levels of GSNOR or GSNOR activity,modulation may be achieved, for example, by administering one or more ofthe disclosed compounds that disrupts or down-regulates GSNOR function,or decreases GSNOR levels. These compounds may be administered withother GSNOR inhibitor agents, such as anti-GSNOR antibodies or antibodyfragments, GSNOR antisense, iRNA, or small molecules, or otherinhibitors, alone or in combination with other agents as described indetail herein.

The present invention provides a method of treating a subject afflictedwith a disorder ameliorated by NO donor therapy. Such a method comprisesadministering to a subject a therapeutically effective amount of a GSNORinhibitor.

The disorders can include pulmonary disorders associated with hypoxemiaand/or smooth muscle constriction in the lungs and airways and/or lunginfection and/or lung inflammation and/or lung injury (e.g., pulmonaryhypertension, ARDS, asthma, pneumonia, pulmonary fibrosis/interstitiallung diseases, cystic fibrosis, COPD); cardiovascular disease and heartdisease (e.g., hypertension, ischemic coronary syndromes,atherosclerosis, heart failure, glaucoma); diseases characterized byangiogenesis (e.g., coronary artery disease); disorders where there isrisk of thrombosis occurring; disorders where there is risk ofrestenosis occurring; inflammatory diseases (e.g., AIDS relateddementia, inflammatory bowel disease (IBD), Crohn's disease, colitis,and psoriasis); functional bowel disorders (e.g., irritable bowelsyndrome (IBS)); diseases where there is risk of apoptosis occurring(e.g., heart failure, atherosclerosis, degenerative neurologicdisorders, arthritis, and liver injury (e.g., drug induced, ischemic oralcoholic)); impotence; sleep apnea; diabetic wound healing; cutaneousinfections; treatment of psoriasis; obesity caused by eating in responseto craving for food; stroke; reperfusion injury (e.g., traumatic muscleinjury in heart or lung or crush injury); and disorders wherepreconditioning of heart or brain for NO protection against subsequentischemic events is beneficial, central nervous system (CNS) disorders(e.g., anxiety, depression, psychosis, and schizophrenia); andinfections caused by bacteria (e.g., tuberculosis, C. difficileinfections, among others).

In one embodiment, the disorder is liver injury. Liver injury caninclude, for example, acute liver toxicity. Acute liver toxicity canresult in acute liver failure. Acute liver failure (ALF) is an uncommonbut potentially lethal drug-related adverse effect that often leads toliver transplantation (LT) or death. Acetoaminophen is the most commoncause of acute liver toxicity and acute liver failure, although acuteliver toxicity can be due to other agents, such as alcohol and otherdrugs. Regardless of whether it occurs as a result of a single overdoseor after repeated supratherapeutic ingestion, the progression ofacetaminophen poisoning can be categorized into four stages: preclinicaltoxic effects (a normal serum alanine aminotransferase concentration),hepatic injury (an elevated alanine aminotransferase concentration),hepatic failure (hepatic injury with hepatic encephalopathy), andrecovery. As long as sufficient glutathione is present, the liver isprotected from injury. Overdoses of acetaminophen (either a single largeingestion or repeated supratherapeutic ingestion) can deplete hepaticglutathione stores and allow liver injury to occur. Compounds of theinvention are capable of treating and/or preventing liver injury and/oracute liver toxicity. In this embodiment, appropriate amounts ofcompounds of the present invention are an amount sufficient to treatand/or prevent liver injury and can be determined without undueexperimentation by preclinical and/or clinical trials. In oneembodiment, the amount to treat is at least 0.001 mg/kg, at least 0.002mg/kg, at least 0.003 mg/kg, at least 0.004 mg/kg, at least 0.005 mg/kg,at least 0.006 mg/kg, at least 0.007 mg/kg, at least 0.008 mg/kg, atleast 0.009 mg/kg, at least 0.01 mg/kg, at least 0.02 mg/kg, at least0.03 mg/kg, at least 0.04 mg/kg, at least 0.05 mg/kg, at least at least0.06 mg/kg, at least 0.07 mg/kg, at least 0.08 mg/kg, at least 0.09mg/kg, at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3 mg/kg, atleast 0.4 mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, at least 0.7mg/kg, at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1 mg/kg, atleast 1.5 mg/kg, at least 2 mg/kg, at least 2.5 mg/kg, at least 3 mg/kg,at least 3.5 mg/kg, at least 4 mg/kg, at least 4.5 mg/kg, at least 5mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9mg/kg, at least 10 mg/kg, at least 15 mg/kg, at least 20 mg/kg, at least30 mg/kg, at least 40 mg/kg, at least 50 mg/kg, at least 60 mg/kg, atleast 70 mg/kg, at least 80 mg/kg, at least 90 mg/kg, at least 100mg/kg. The dosing can be hourly, four times, twice, or once daily, orfour times, twice, or once per week, or weekly, or every other week,every third week, or monthly.

In one embodiment, the disorder is trauma (including surgery andthermal), infectious, toxic, aging, and ischemic damage to organs ofknown regenerative capacity, such as skin, gastric mucosa, airwayepithelial and cartilaginous structures, liver, neuronal structures suchas the spinal cord, bone marrow and bone. We have shown that inhibitionof GSNOR by the use of highly specific small molecules treats, repairs,and promotes regeneration of mammalian tissue. By way of example, smallmolecule inhibitors are effective in treating, and promoting repair andregeneration of mammalian lung tissue damaged by instillation of achemical agent known to cause severe lung injury (porcine pancreaticelastase) (Blonder et al., ATS 2011 abstract reference). In thisembodiment, appropriate amounts of compounds of the present inventionare an amount sufficient to regenerate tissue/organs and can bedetermined without undue experimentation by preclinical and/or clinicaltrials.

In one embodiment the disorder is trauma (including surgery andthermal), infectious, toxic, aging, and ischemic damage to organs of notcommonly known to have regenerative capacity. Examples includeregeneration of: the heart, the lung, the kidney, the central nervoussystem, the peripheral nervous system, peripheral vascular tissue,liver, pancreas, adrenal gland, thyroid, testes, ovary, retina, tongue,bone, bladder, esophagus, larynx, thymus, spleen, cartilaginousstructures of the head, and cartilaginous structures of the joints. Inthis embodiment, appropriate amounts of compounds of the presentinvention are an amount sufficient to regenerate tissue/organs and canbe determined without undue experimentation by preclinical and/orclinical trials.

In one embodiment ex and in vivo implantation and regeneration of organsand structures, including stem cells. In this embodiment, appropriateamounts of compounds of the present invention are an amount sufficientto regenerate tissue/organs and can be determined without undueexperimentation by preclinical and/or clinical trials.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, or a prodrug, stereoisomer,metabolite, or N-oxide thereof, can be administered in combination withan NO donor. An NO donor donates nitric oxide or a related redox speciesand more generally provides nitric oxide bioactivity, that is activitywhich is identified with nitric oxide, e.g., vasorelaxation orstimulation or inhibition of a receptor protein, e.g., ras protein,adrenergic receptor, NFκB. NO donors including S-nitroso, O-nitroso,C-nitroso, and N-nitroso compounds and nitro derivatives thereof andmetal NO complexes, but not excluding other NO bioactivity generatingcompounds, useful herein are described in “Methods in Nitric OxideResearch,” Feelisch et al. eds., pages 71-115 (J. S., John Wiley & Sons,New York, 1996), which is incorporated herein by reference. NO donorswhich are C-nitroso compounds where nitroso is attached to a tertiarycarbon which are useful herein include those described in U.S. Pat. No.6,359,182 and in WO 02/34705. Examples of S-nitroso compounds, includingS-nitrosothiols useful herein, include, for example,S-nitrosoglutathione, S-nitroso-N-acetylpenicillamine,S-nitroso-cysteine and ethyl ester thereof, S-nitroso cysteinyl glycine,S-nitroso-gamma-methyl-L-homocysteine, S-nitroso-L-homocysteine,S-nitroso-gamma-thio-L-leucine, S-nitroso-delta-thio-L-leucine, andS-nitrosoalbumin. Examples of other NO donors useful herein are sodiumnitroprusside (nipride), ethyl nitrite, isosorbide, nitroglycerin, SIN 1which is molsidomine, furoxamines, N-hydroxy (N-nitrosamine), andperfluorocarbons that have been saturated with NO or a hydrophobic NOdonor.

The combination of a GSNOR inhibitor with R(+) enantiomer of amlodipine,a known NO releaser (Zhang at al., J. Cardiovasc. Pharm. 39: 208-214(2002)) is also an embodiment of the present invention.

The present invention also provides a method of treating a subjectafflicted with pathologically proliferating cells where the methodcomprises administering to said subject a therapeutically effectiveamount of an inhibitor of GSNOR. The inhibitors of GSNOR are thecompounds as defined above, or a pharmaceutically acceptable saltthereof, or a stereoisomer, prodrug, metabolite, or N-oxide thereof, incombination with a pharmaceutically acceptable carrier. Treatment iscontinued as long as symptoms and/or pathology ameliorate.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating microbes. The microbes involved can bethose where GSNOR is expressed to protect the microbe from nitrosativestress or where a host cell infected with the microbe expresses theenzyme, thereby protecting the microbe from nitrosative stress. The term“pathologically proliferating microbes” is used herein to meanpathologic microorganisms including, but not limited to, pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367. The term “host cells infected withpathologic microbes” includes not only mammalian cells infected withpathologic viruses but also mammalian cells containing intracellularbacteria or protozoa, e.g., macrophages containing Mycobacteriumtuberculosis, Mycobacterium leper (leprosy), or Salmonella typhi(typhoid fever).

In another embodiment, the pathologically proliferating cells can bepathologic helminths. The term “pathologic helminths” is used herein torefer to pathologic nematodes, pathologic trematodes and pathologiccestodes. More detail on the applicable helminths is set forth at column12 of U.S. Pat. No. 6,057,367.

In another embodiment, the pathologically proliferating cells can bepathologically proliferating mammalian cells. The term “pathologicallyproliferating mammalian cells” as used herein means cells of the mammalthat grow in size or number in said mammal so as to cause a deleteriouseffect in the mammal or its organs. The term includes, for example, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatichypertrophy, the pathologically proliferating cells causing myocardialhypertrophy, and proliferating cells at inflammatory sites such assynovial cells in arthritis or cells associated with a cellproliferation disorder.

As used herein, the term “cell proliferative disorder” refers toconditions in which the unregulated and/or abnormal growth of cells canlead to the development of an unwanted condition or disease, which canbe cancerous or non-cancerous, for example a psoriatic condition. Asused herein, the term “psoriatic condition” refers to disordersinvolving keratinocyte hyperproliferation, inflammatory cellinfiltration, and cytokine alteration. The cell proliferative disordercan be a precancerous condition or cancer. The cancer can be primarycancer or metastatic cancer, or both.

As used herein, the term “cancer” includes solid tumors, such as lung,breast, colon, ovarian, pancreas, prostate, adenocarcinoma, squamouscarcinoma, sarcoma, malignant glioma, leiomyosarcoma, hepatoma, head andneck cancer, malignant melanoma, non-melanoma skin cancers, as well ashematologic tumors and/or malignancies, such as leukemia, childhoodleukemia and lymphomas, multiple myeloma, Hodgkin's disease, lymphomasof lymphocytic and cutaneous origin, acute and chronic leukemia such asacute lymphoblastic, acute myelocytic, or chronic myelocytic leukemia,plasma cell neoplasm, lymphoid neoplasm, and cancers associated withAIDS.

In addition to psoriatic conditions, the types of proliferative diseaseswhich may be treated using the compositions of the present invention areepidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneoushemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas,myofibromatosis, osteoplastic tumors, and other dysplastic masses, andthe like. In one embodiment, proliferative diseases include dysplasiasand disorders of the like.

In one embodiment, treating cancer comprises a reduction in tumor size,decrease in tumor number, a delay of tumor growth, decrease inmetastatic lesions in other tissues or organs distant from the primarytumor site, an improvement in the survival of patients, or animprovement in the quality of patient life, or at least two of theabove.

In another embodiment, treating a cell proliferative disorder comprisesa reduction in the rate of cellular proliferation, reduction in theproportion of proliferating cells, a decrease in size of an area or zoneof cellular proliferation, or a decrease in the number or proportion ofcells having an abnormal appearance or morphology, or at least two ofthe above.

In yet another embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof can beadministered in combination with a second chemotherapeutic agent. In afurther embodiment, the second chemotherapeutic agent is selected fromthe group consisting of tamoxifen, raloxifene, anastrozole, exemestane,letrozole, cisplatin, carboplatin, paclitaxel, cyclophosphamide,lovastatin, minosine, gemcitabine, araC, 5-fluorouracil, methotrexate,docetaxel, goserelin, vincristin, vinblastin, nocodazole, teniposide,etoposide, epothilone, navelbine, camptothecin, daunonibicin,dactinomycin, mitoxantrone, amsacrine, doxorubicin, epirubicin,idarubicin imatanib, gefitinib, erlotinib, sorafenib, sunitinib malate,trastuzumab, rituximab, cetuximab, and bevacizumab.

In one embodiment, the compounds of the present invention or apharmaceutically acceptable salt thereof, a stereoisomer thereof, aprodrug thereof, a metabolite thereof, or an N-oxide thereof, can beadministered in combination with an agent that imposes nitrosative oroxidative stress. Agents for selectively imposing nitrosative stress toinhibit proliferation of pathologically proliferating cells incombination therapy with GSNOR inhibitors herein and dosages and routesof administration therefor include those disclosed in U.S. Pat. No.6,057,367, which is incorporated herein. Supplemental agents forimposing oxidative stress (i.e., agents that increase GSSG (oxidizedglutathione) over GSH (glutathione) ratio or NAD(P) over NAD(P)H ratioor increase thiobarbituric acid derivatives) in combination therapy withGSNOR inhibitors herein include, for example, L-buthionine-S-sulfoximine(BSO), glutathione reductase inhibitors (e.g., BCNU), inhibitors oruncouplers of mitochondrial respiration, and drugs that increasereactive oxygen species (ROS), e.g., adriamycin, in standard dosageswith standard routes of administration.

GSNOR inhibitors may also be co-administered with a phosphodiesteraseinhibitor (e.g., rolipram, cilomilast, roflumilast, Viagra® (sildenifilcitrate), Cialis® (tadalafil), Levitra® (vardenifil), etc.), aβ-agonist, a steroid, an anti-muscarinics, or a leukotriene antagonist(LTD-4). Those skilled in the art can readily determine the appropriatetherapeutically effective amount depending on the disorder to beameliorated.

GSNOR inhibitors may be used as a means to improve β-adrenergicsignaling. In particular, inhibitors of GSNOR alone or in combinationwith β-agonists could be used to treat or protect against heart failure,or other vascular disorders such as hypertension and asthma. GSNORinhibitors can also be used to modulate G protein coupled receptors(GPCRs) by potentiating Gs G-protein, leading to smooth musclerelaxation (e.g., airway and blood vessels), and by attenuating GqG-protein, and thereby preventing smooth muscle contraction (e.g., inairway and blood vessels).

The therapeutically effective amount for the treatment of a subjectafflicted with a disorder ameliorated by NO donor therapy is the GSNORinhibiting amount in vivo that causes amelioration of the disorder beingtreated or protects against a risk associated with the disorder. Forexample, for asthma, a therapeutically effective amount is abronchodilating effective amount; for cystic fibrosis, a therapeuticallyeffective amount is an airway obstruction ameliorating effective amount;for ARDS, a therapeutically effective amount is a hypoxemia amelioratingeffective amount; for heart disease, a therapeutically effective amountis an angina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective amount is an erection attaining or sustaining effectiveamount; for obesity, a therapeutically effective amount is a satietycausing effective amount; for stroke, a therapeutically effective amountis a blood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by troponin or CPK.

The therapeutically effective amount for the treatment of a subjectafflicted with pathologically proliferating cells means a GSNORinhibiting amount in vivo which is an antiproliferative effectiveamount. Such antiproliferative effective amount as used herein means anamount causing reduction in rate of proliferation of at least about 20%,at least about 10%, at least about 5%, or at least about 1%.

I. Uses in an Apparatus

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a stereoisomer, prodrug, metabolite, or N-oxidethereof, can be applied to various apparatus in circumstances when thepresence of such compounds would be beneficial. Such apparatus can beany device or container, for example, implantable devices in which acompound of the invention can be used to coat a surgical mesh orcardiovascular stent prior to implantation in a patient. The compoundsof the invention can also be applied to various apparatus for in vitroassay purposes or for culturing cells.

The compounds of the present invention or a pharmaceutically acceptablesalt thereof, or a stereoisomer, a prodrug, a metabolite, or an N-oxidethereof, can also be used as an agent for the development, isolation orpurification of binding partners to compounds of the invention, such asantibodies, natural ligands, and the like. Those skilled in the art canreadily determine related uses for the compounds of the presentinvention.

EXAMPLES

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

Example 1-32 list representative novel napthalene analogs of Formula 1useful as GSNOR inhibitors of the invention. Synthetic methods that canbe used to prepare each compound are described in Examples 1-32.Supporting mass spectrometry data and/or proton NMR data for eachcompound is also included in the Examples. Synthetic details forcorresponding Intermediates are detailed in Example 34 .

Example 1 3-chloro-4-(6-hydroxynaphthalen-2-yl)benzoic acid

A mixture of 6-bromonaphthalen-2-ol (500 mg, 2.20 mmol),4-borono-3-chlorobenzoic acid (449 mg, 2.20 mmol), K₃PO₄ (1.40 g, 6.60mmol) and Pd(PPh₃)₄ (200 mg, 0.170 mmol) in DMF (15 mL) and water (3 mL)was stirred under N₂ atmosphere at 80° C. for 3 hours. The resultingmixture was cooled to room temperature and diluted with water (50 mL)and extracted with ethyl acetate (50 mL×3). The aqueous phase wasacidified with aqueous HCl until pH=2 and extracted with ethyl acetate(50 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was purified byprep. HPLC (0.1% TFA as additive) to give compound Example 1 (59 mg,yield 9%) as an off-white solid. ¹H NMR (CD₃OD 400 MHz): δ 8.15 (d,J=1.6 Hz, 1H), 8.04 (dd, J=8.0, 1.6 Hz, 1H), 7.85 (s, 1H), 7.80 (d,J=8.8 Hz, 1H), 7.74 (d, J=8.4 Hz, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.51 (dd,J=8.4, 1.6 Hz, 1H), 7.18 (d, J=2.0 Hz, 1H), 7.14 (dd, J=8.8, 2.4 Hz,1H). MS (ESI): m/z 297.0 [M−1]⁻.

Example 2 3-fluoro-4-(6-hydroxynaphthalen-2-yl)benzoic acid

6-Bromonaphthalen-2-ol (1 g, 4.48 mmol) was mixed with 880 mg of2-fluoro-4-(methoxycarbonyl)phenylboronic acid (4.48 mmol), 1.2 g ofK₂CO₃ (9.23 mmol), 120 mg of Pd(PPh₃)₄ (2.5 mol %), and then suspendedin 17 mL of 1,4-Dioxane and 4 mL of H₂O. The mixture was degassed 3×with argon and then heated to reflux while stirring for 1 hour. 4N NaOH(5 mL) was then added and stirred at room temperature for 2 hours, backextracted with EtOAc, and filtered through Celite. The basic aqueoussolution was then acidified to pH=4.3 and the solids were filtered togive about 950 mg of off-white solid. This was triturated in 8 mL DCMand 2 mL EtOAc and filtered to yield about 830 mg of off-white Example 2(65.8% overall yield). ¹H-NMR (DMSO-d6 500 MHz): δ 13.31 (bs, 1H), 9.97(bs, 1H), 8.05 (s, 1H), 7.90 (m, 2H), 7.80 (m, 3H), 7.64 (dt, 1H), 7.19(m, 2H); MS (ESI): m/z 281.53 [M−1]⁻.

Example 3 4-(6-hydroxynaphthalen-2-yl)-3-methoxybenzoic acid

A mixture of 6-bromonaphthalen-2-ol (124 mg, 0.559 mmol),4-(dihydroxyboryl)-3-methoxybenzoic acid (100 mg, 0.510 mmol) and K₂CO₃(211 mg, 1.53 mmol) in DME/H₂O (7 mL/2 mL) was degassed three timesunder N₂. Then PdCl₂(dppf) (37 mg, 0.0559 mmol) was added and themixture was heated to 110° C. for 3 hours. Followed theworkup/purification procedure described in Example 1 to give Example 3(60 mg, yield 37%) as a yellow solid. ¹H NMR (DMSO 400 MHz): δ 13.00(brs, 1H), 9.78 (brs, 1H), 7.89 (s, 1H), 7.80 (d, J=8.8 Hz, 1H) 7.70 (d,J=8.4 Hz, 1H), 7.65-7.57 (m, 2H), 7.55-7.45 (m, 2H), 7.10-7.02 (m, 2H),3.85 (s, 3H). MS (ESI): m/z 293.0 [M−H]⁻.

Example 4 5-(6-hydroxynaphthalen-2-yl)picolinic acid

Step 1: Synthesis of methyl 5-(6-hydroxynaphthalen-2-yl)picolinate: Amixture of 6-bromonaphthalen-2-ol (250 mg, 1.13 mmol), methyl5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)picolinate (355 mg, 1.35mmol), Pd(dppf)Cl₂ (82 mg, 0.112 mmol) and sodium carbonate (263 mg,2.48 mmol) in DME/water (3 mL/1 mL) was heated to 100° C. by microwavefor 1 h. Then the mixture was partitioned with water (10 mL) and EA (20mL). The precipitate was filtered off. The organic phase was separated,washed with brine (15 mL), dried over Na₂SO₄ and concentrated to givethe product (180 mg, 57%) as a brown solid.

Step 2: Synthesis of Example 4: Methyl5-(6-hydroxynaphthalen-2-yl)picolinate (180 mg, 0.66 mmol) was dissolvedin THF/water (3 mL/1 mL), then sodium hydroxide (78 mg, 1.9 mmol) wasadded. The mixture was refluxed for 3 hours, concentrated and purifiedby prep-HPLC to afford Example 4 (55 mg, 31.4%) as a yellow powder. ¹HNMR (DMSO-d₆ 500 MHz TMS): 9.95 (s, 1H), 9.13 (s, 1H), 8.36 (dd, J=2.0Hz, J=8.0 Hz, 1H), 8.29 (s, 1H), 8.13 (d, J=8.5 Hz, 1H), 7.89 (d, J=8.5Hz, 1H), 7.85 (s, 2H), 7.18˜7.14 (m, 2H); MS (ESI): m/z 266.1 [M+1]⁺.

Example 5 3-(dimethylamino)-4-(6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl3-amino-4-(6-methoxynaphthalen-2-yl)benzoate: A mixture of2-bromo-6-methoxynaphthalene (10.0 g, 51.3 mmol).2-amino-4-(methoxycarbonyl)phenylboronic acid (10 g) and K₂CO₃ (18.0 g,133 mmol) in CH₃OCH₂CH₂OH/H₂O (140 mL/40 mL) was degassed three timesunder N₂. Then Pd(dppf)Cl₂ (3.10 g, 4.23 mmol) was added quickly and themixture was heated to 60° C. for 1 hour. The mixture was diluted withH₂O (200 mL) and EtOAc (500 mL) and filtered. The filtrate was separatedand the aqueous layer was extracted with EtOAc (100 mL×3), the combinedorganic layers were washed with brine (200 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby column chromatography on silica gel (PE/EtOAc=10/1) to give theproduct (4.20 g) as an off-white solid.

Step 2: Synthesis of methyl3-(dimethylamino)-4-(6-methoxynaphthalen-2-yl)benzoate: To a solution ofthe above product (1.00 g, 3.30 mmol) in MeOH/CH₂Cl₂ (10 mL/20 mL) wasadded aqueous HCHO (37% aqueous, 5 mL), followed by NaBH₃CN (820 mg,13.0 mmol) and ZnCl₂ (900 mg, 6.50 mmol) at 0° C. The mixture wasstirred at 30° C. for 10 hours. The mixture was quenched with ice water(50 mL), the aqueous layer was extracted with CH₂Cl₂ (30 mL×3), thecombined organic layer was washed with brine (50 mL), dried overanhydrous Na₂SO₄ and concentrated in vacuo to give the product (1.00 g,yield 90%).

Step 3: Synthesis of methyl3-(dimethylamino)-4-(6-hydroxynaphthalen-2-yl)benzoate: To a solution ofthe above product (450 mg, 1.34 mmol) in anhydrous CH₂Cl₂ (10 mL) wasadded BBr₃ (0.6 mL 6.7 mmol) dropwise at 0° C. The mixture was stirredat 0° C. for 3 hours, then quenched with ice water (20 mL). The aqueouslayer was extracted with EtOAc (20 mL×3), the combined organic layerswashed with brine (50 mL), dried over anhydrous Na₂SO₄ and concentratedin vacuo to give the product (340 mg, yield 79%) as an off-white solid.

Step 4: Synthesis of3-(dimethylamino)-4-(6-hydroxynaphthalen-2-yl)benzoic acid: To asolution of the above product (340 mg, 1.10 mmol) in THF/MeOH (8 mL/4mL) was added aqueous LiOH (1 M, 4 mL). The mixture was stirred at 30°C. for 16 hours. The mixture was acidified with HCl (10%) to pH 6 andextracted with EtOAc (30 mL×3). The combined organic layers were washedwith brine (20 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by prep-TLC (PE/EtOAc=1/1) togive Example 5 (25 mg, yield 8%) as a yellow solid. ¹H NMR (MeOD 400MHz): δ 7.91 (s, 1H), 7.80-7.70 (m, 2H), 7.70-7.63 (m, 3H), 7.15 (d,J=2.4 Hz, 1H), 7.10 (dd, J=8.8, 2.4 Hz, 2H), 2.65 (s, 6H). MS (ESI): m/z308.1 [M+H]⁺.

Example 6 3-cyano-4-(6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl3-cyano-4-(6-methoxynaphthalen-2-yl)benzoate: A mixture of methyl3-cyano-4-(trifluoromethylsulfonyloxy)benzoate (Intermediate 1) (550 mg,1.78 mmol), 6-methoxy-2-naphthaleneboronic acid (720 mg, 3.56 mmol),KOAc (700 mg, 7.12 mmol) and Pd(dppf)Cl₂ (100 mg, 0.122 mmol) in DME (10mL), EtOH (10 mL) and water (10 mL) was stirred at 90° C. for 2 hoursunder N₂ atmosphere. The resulting mixture was cooled to roomtemperature, suspended in water (50 mL) and extracted with ethyl acetate(50 mL×3). The combined organic layers were dried over anhydrous Na₂SO₄and concentrated in vacuo, then purified by column chromatography onsilica gel (PE/EtOAc=20/1) to give the product (500 mg, yield: 89%) asan off-white solid.

Step 2: Synthesis of 3-cyano-4-(6-hydroxynaphthalen-2-yl)benzoic acid: Amixture of the above product (200 mg, 0.631 mmol) and BBr₃ (2 mL, 21.2mmol) in anhydrous DCM (20 mL) was stirred at 25° C. overnight. Theresulting mixture was quenched with water (50 mL) and extracted withethyl acetate (50 mL×3). The combined organic layers were dried overanhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified byprep-HPLC (0.1% TFA as additive) to give Example 6 (55 mg, yield 30%) asa off-white solid. ¹H NMR (DMSO-d₆ 400 MHz): δ 13.54 (brs, 1H), 10.00(brs, 1H), 8.37 (d, J=1.6 Hz, 1H), 8.26 (dd, J=8.4, 2.0 Hz, 1H), 8.06(d, J=1.2 Hz, 1H), 7.92-7.82 (m, 3H), 7.63 (dd, J=8.4, 2.0 Hz, 1H),7.22-7.12 (m, 2H). MS (ESI): m/z 290.0 [M+H]⁺, 312.1 [M+Na]⁺.

Example 7 6-(6-hydroxynaphthalen-2-yl)nicotinic acid

Step 1: Synthesis of methyl 6-(6-hydroxynaphthalen-2-yl)nicotinate: Amixture of 6-hydroxynaphthalen-2-ylboronic acid (250 mg, 1.33 mmol),methyl 6-bromonicotinate (260 mg, 1.21 mmol), Pd(dppf)Cl₂ (90 mg, 0.12mmol) and sodium carbonate (205 mg, 2.41 mmol) in DME/water (3 mL/1 mL)was heated to 120° C. by microwave for 1 h. Then the mixture waspartitioned with water (10 mL) and EA (20 mL). The precipitate wasfiltered off. The organic phase was separated, washed with brine (15mL), dried over Na₂SO₄ and concentrated to afford product (crude, 380mg) as a brown oil.

Step 2: Synthesis of 6-(6-hydroxynaphthalen-2-yl)nicotinic acid: methyl6-(6-hydroxynaphthalen-2-yl)nicotinate (380 mg, 1.36 mmol) was dissolvedin THF/water (6 mL/2 mL), then sodium hydroxide (164 mg, 4.09 mmol) wasadded. The mixture solution was heated to reflux for 3 hours,concentrated and purified by prep-HPLC to afford Example 7 (20 mg, 5.5%)as a powder. ¹H NMR (MeOD-d₄ 500 MHz): 9.21 (s, 1H), 8.55 (dd, J=2.0 Hz,J=8.5 Hz, 1H), 8.51 (s, 1H), 8.19 (d, J=8.0 Hz, 1H), 8.09 (dd, J=2.0 Hz,J=9.0 Hz, 1H), 7.91 (d, J=8.0 Hz, 1H), 7.82 (d, J=9.0 Hz, 1H), 7.19˜7.17(m, 2H); MS (ESI): m/z 266.1 [M+1]⁺.

Example 8 5-(6-hydroxynaphthalen-2-yl)pyrazine-2-carboxylic acid

Followed the two step procedure described for Example 7 starting from6-hydroxynaphthalen-2-ylboronic acid and methyl5-chloropyrazine-2-carboxylate. ¹H-NMR (MeOD-d₄ 500 MHz): 9.31 (s, 2H),8.64 (s, 1H), 8.19˜8.21 (d, J=9.0 Hz, 1H), 7.90˜7.91 (d, J=8.5 Hz, 1H),7.80˜7.82 (d, J=8.5 Hz, 1H), 7.15˜7.18 (t, J=2.0, 10.0 Hz, 2H). MS(ESI): m/z=267.0 [M+1]⁺.

Example 9 2-(6-hydroxynaphthalen-2-yl)pyrimidine-5-carboxylic acid

Followed procedure described in Step 1 of Example 7 starting from6-hydroxynaphthalen-2-ylboronic acid and 2-chloropyrimidine-5-carboxylicacid with modifications: the solvent was dioxane/water (6/1), and thereaction was heated to 120° C. by microwave for 0.5 h. Purified byprep-HPLC. ¹H-NMR (DMSO-d₆ 500 MHz): 13.70 (brs, 1H), 10.12 (s, 1H),9.29 (s, 2H), 8.97 (s, 1H), 8.42˜8.44 (d, J=9.0 Hz, 1H), 8.00˜8.01 (d,J=8.5 Hz, 1H), 7.82˜7.84 (d, J=9.0 Hz, 1H), 7.20 (s, 1H), 7.15˜7.18 (dd,J=2.0, 9.0 Hz, 2H). MS (ESI): m/z=267.0 [M+1]⁺.

Example 10 4-(6-hydroxynaphthalen-2-yl)-3-morpholinobenzoic acid

Step 1: Synthesis of methyl4-(6-methoxynaphthalen-2-yl)-3-morpholinobenzoate: To a solution ofmethyl 3-amino-4-(6-methoxynaphthalen-2-yl)benzoate (1.00 g, 3.30 mmol)(see Step 1 of Example 5) and DIPEA (1.70 g, 13.2 mmol) in anhydroustoluene (20 mL) containing catalytic amount of KI (100 mg, 0.660 mmol)was added 2,2′-dibromodiethyl ether (1.50 g, 6.60 mmol). The mixture washeated to 110° C. for 3 days. The mixture was concentrated in vacuo andthe residue was purified by column chromatography on silica gel(PE/EtOAc=10/1) to give the product (1.00 g, yield 81%).

Step 2: Synthesis of methyl4-(6-hydroxynaphthalen-2-yl)-3-morpholinobenzoate: Followed the BBr₃deprotection procedure described in Step 3 of Example 5.

Step 3: 4-(6-hydroxynaphthalen-2-yl)-3-morpholinobenzoic acid: Followedprocedure described in Step 4 of Example 5, where reaction was run for20 h at 25° C. The prep-HPLC purification (0.1% TFA as additive) gaveExample 10 (54 mg, two-step yield 5.8%) as an off-white solid. ¹H NMR(DMSO 400 MHz): δ 9.82 (brs, 1H), 8.00 (s, 1H) 7.80 (m, 2H), 7.74-7.60(m, 3H), 7.39 (d, J=8.0 Hz, 1H), 7.13 (d, J=2.0 Hz, 1H), 7.08 (dd,J=8.8, 2.4 Hz, 1H), 3.52-3.47 (m, 4H), 2.80-2.72 (m, 4H). MS (ESI): m/z349.9 [M+H]⁺.

Example 11 6-(6-hydroxynaphthalen-2-yl)pyridazine-3-carboxylic acid

Followed the two step procedure described for Example 7 starting from6-hydroxynaphthalen-2-ylboronic acid and methyl6-chloropyridazine-3-carboxylate. ¹H-NMR (DMSO-d₆ 500 MHz): 10.16 (s,1H), 8.71 (d, J=5.0 Hz, 1H), 8.51˜8.48 (m, 1H), 8.30˜8.23 (m, 2H),7.97˜7.86 (m, 2H), 7.23˜7.19 (m, 2H). MS (ESI): m/z 267.0 [M+1]⁺.

Example 12 4-(1-bromo-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl4-(1-bromo-6-methoxynaphthalen-2-yl)benzoate: Followed the couplingprocedure described for Example 1, starting from1-bromo-6-methoxynaphthalen-2-yl trifluoromethanesulfonate (Intermediate2) and 4-(methoxycarbonyl)phenylboronic acid with modifications: thebase used was Na₂CO₃, the solvent was toluene/EtOH (4/1), and thereaction was heated to 80° C. for 3 h. The crude product was purified bysilica gel column (PE/EtOAc=300/1 to 100/1), then washed with EtOAc (10mL×5) to give the product (980 mg, yield 45%) as a white solid.

Step 2: Synthesis of 4-(1-bromo-6-hydroxynaphthalen-2-yl)benzoic acid:To a solution of above product (200 mg, 0.539 mmol) in anhydrous DCM (10mL) was added BBr₃ (0.26 mL, 2.70 mmol, d=2.64 g/mL) dropwise at 0° C.The resulting mixture was stirred at 25° C. for 2 hours. The reactionmixture was quenched with H₂O (30 mL), then extracted with DCM (20mL×3). The combined organic layers were washed with brine (20 mL), driedover anhydrous Na₂SO₄, filtered and concentrated. The crude product waspurified by prep-HPLC (0.1% TFA as additive). Most of CH₃CN was removedunder reduced pressure and the remaining solvent was removed bylyophilization to give Example 12 (70 mg, yield: 39%) as a white solid.¹H NMR (DMSO-d₆ 400 MHz): δ 13.05 (brs, 1H), 10.16 (brs, 1H), 8.16 (d,J=9.2 Hz, 1H), 8.04 (d, J=8.4 Hz, 2H), 7.82 (d, J=8.4 Hz, 1H), 7.57 (d,J=8.4 Hz, 2H), 7.37 (d, J=8.4 Hz, 1H), 7.32-7.21 (m, 2H). MS (ESI): m/z341.0 [M−H]⁻.

Example 13 4-(6-hydroxy-1-methylnaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl4-(6-methoxy-1-methylnaphthalen-2-yl)benzoate: A mixture of methyl4-(1-bromo-6-methoxynaphthalen-2-yl)benzoate (300 mg, 0.811 mmol) (seeExample 12 step 1), MeZnCl (1.2 mL, 2.40 mmol, 2M in THF) and Pd(PPh₃)₄(93.8 mg, 0.0811 mmol) in anhydrous THF (10 mL) was stirred at 60° C.under N₂ atmosphere for 16 hours. After cooling to room temperature, themixture was quenched with saturated aqueous NH₄Cl (20 mL), extractedwith EtOAc (20 mL×3). The combined organic layers were washed with H₂O(20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to give the crude product. The crudeproduct was purified by silica gel column (PE/EtOAc=300/1 to 200/1) togive the product (210 mg, yield 85%) as a solid.

Step 2: Synthesis of methyl4-(6-hydroxy-1-methylnaphthalen-2-yl)benzoate: Followed the BBr₃deprotection procedure described for Step 2 of Example 12, except thecrude product was taken forward without purification.

Step 3: Synthesis of Example 13: To a solution of the above product (200mg, crude from above) in MeOH (8 mL) was added aqueous NaOH (8 mL, 2M).The mixture was stirred at 25° C. for 16 hours. To the reaction mixturewas added H₂O (30 mL), extracted with EtOAc (15 mL×2) and discarded; theaqueous layer was acidified with 1N HCl to pH=1˜2, extracted with EtOAc(15 mL×3). The combined organic layers were washed with brine (20 mL),dried over anhydrous Na₂SO₄, filtered and concentrated. Purification byprep-HPLC (0.1% TFA as additive); then most of CH₃CN was removed invacuo followed by lyophilization to give Example 13 (25 mg, 2-step yield13%) as an off-white solid. ¹H NMR (MeOD 400 MHz): δ 8.09 (d, J=8.0 Hz,2H), 7.99 (d, J=8.8 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.45 (d, J=8.0 Hz,2H), 7.25 (d, J=8.4 Hz, 1H), 7.20-7.10 (m, 2H), 2.53 (s, 3H). MS (ESI):m/z 300.9 [M+Na]⁺.

Example 14 5-(6-hydroxynaphthalen-2-yl)pyrimidine-2-carboxylic acid

Followed the two step procedure described for Example 7 starting from6-hydroxynaphthalen-2-yl boronic acid and methyl5-bromopyrimidine-2-carboxylate. ¹H-NMR (DMSO-d₆ 500 MHz): 9.39 (s, 2H),8.38 (s, 1H), 7.87˜7.91 (t, J=8.5, 12.0 Hz, 3H), 7.17˜7.20 (t, J=5.5,10.5 Hz, 2H). MS (ESI): m/z=267.0 [M+1]⁺.

Example 15 4-(1-cyano-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl4-(1-cyano-6-methoxynaphthalen-2-yl)benzoate: To a solution of methyl4-(1-bromo-6-methoxynaphthalen-2-yl)benzoate (300 mg, 0.811 mmol) (seeExample 12 step 1) in DMF (10 mL) was added Zn(CN)₂ (191 mg, 1.62 mmol)and Pd(PPh₃)₄ (93.8 mg, 0.0811 mmol). The resulting mixture was stirredat 120° C. under N₂ atmosphere for 16 hours. After cooling to roomtemperature, the mixture was filtered and the filtrate was diluted withEtOAc (60 mL), washed with H₂O (20 mL×3) and brine (20 mL), dried overanhydrous Na₂SO₄, filtered and concentrated in vacuo. Purification bysilica gel column (PE/EtOAc=200/1 to 50/1) gave the product (210 mg,yield 82%) as a solid.

Step 2: Synthesis of methyl4-(1-cyano-6-hydroxynaphthalen-2-yl)benzoate: Followed the BBr3deprotection procedure described for Step 2 of Example 12, except thecrude product was taken forward without purification.

Step 3: Synthesis of Example 15: To a solution of above product (160 mg,crude from above) in THF (16 mL) was added aqueous LiOH (4 mL, 2M). Themixture was stirred at 25° C. for 16 hours. To the reaction mixture wasadded H₂O (30 mL), extracted with EtOAc and discarded; then aqueouslayer acidified with 1N HCl to pH=5˜6, extracted with EtOAc (15 mL×3),washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated. The crude product was washed with EtOAc (10 mL) to giveExample 15 (65 mg, 2-step yield 34%) as a pale-yellow solid. ¹H NMR(DMSO-d₆ 400 MHz): δ 8.17 (d, J=8.8 Hz, 1H), 8.12-8.05 (m, 3H), 7.79 (d,J=8.4 Hz, 2H), 7.65 (d, J=8.8 Hz, 1H), 7.42 (dd, J=8.8 Hz, 2.4 Hz, 1H),7.37 (d, J=2.4 Hz, 1H). MS (ESI): m/z 288.0 [M−H]⁻.

Example 16 4-(6-hydroxy-3-methoxynaphthalen-2-yl)benzoic acid

Followed the coupling procedure described for Example 3, starting from6-bromo-7-methoxynaphthalen-2-ol (Intermediate 3) and 4-boronobenzoicacid with a purification by prep-HPLC (0.1% TFA as additive) to giveExample 16 (93 mg, yield 36%) as an off-white solid. ¹H NMR (DMSO 400MHZ): δ 9.72 (brs, 1H), 7.92 (d, J=8.4 Hz, 2H), 7.68-7.63 (m, 2H), 7.60(d, J=8.4 Hz, 2H), 7.17 (s, 1H), 7.02 (d, J=2.0 Hz, 1H), 6.87 (dd,J=8.8, 2.0 Hz, 1H), 3.79 (s, 3H). MS (ESI): m/z 293.1 [M+H]⁺.

Example 17 4-(1-chloro-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of 4-(1-chloro-6-methoxynaphthalen-2-yl)benzoic acid:Followed the coupling procedure described for Example 3, starting from1-chloro-6-methoxynaphthalen-2-yl trifluoromethanesulfonate(Intermediate 4, 1 equiv) and 4-boronobenzoic acid (1.2 equiv), wherethe mixture was stirred at 80° C. for 2 hours. The crude product wasobtained after workup and was taken on without purification.

Step 2: Synthesis of Example 17: Followed the deprotection methoddescribed in Step 2 of Example 12 to give Example 17 (50 mg, yield 28%)as an off-white solid. ¹H NMR (MeOD 400 MHz): δ 8.22 (d, J=9.2 Hz, 1H),8.10 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.4 Hz, 1H), 7.58 (d, J=8.4 Hz, 2H),7.36 (d, J=8.4 Hz, 1H), 7.23 (dd, J=9.2 Hz, 2.4 Hz, 1H), 7.18 (d, J=2.4Hz, 1H). MS (ESI): m/z 297.0 [M−H]⁻.

Example 18 6-(4-(1H-tetrazol-5-yl)phenyl)naphthalen-2-ol

Followed the coupling procedure described in step 1 of Example 7,starting from 6-bromonaphthalen-2-ol and4-(1H-tetrazol-5-yl)phenylboronic acid where the reaction was run (EtOHas a co-solvent and K₂CO₃ as the base) for 1.5 h at 150° C. The reactionmixture was filtered through Celite and washed with 1N NaOH. The motherliquor was then acidified to a pH of about 4 with conc. HCl. Theresulting solid was filtered, washed with H₂O, and dried in vacuo toyield the crude product. This was taken up in hot ethanol, treated withactivated charcoal, and allowed to recrystallize from the EtOH. IsolatedExample 18 by filtration (108 mg, 17%). ¹H NMR (DMSO-d₆ 500 MHz): δ 9.90(s, 1H), 8.23 (s, 1H), 8.14 (d, 2H), 8.05 (d, 2H), 7.90 (d, 1H), 7.82(s, 2H), 7.17 (m, 2H). MS (ESI): m/z 289.02 [M+1]⁺.

Example 19 6-(1H-benzo[d][1,2,3]triazol-6-yl)naphthalen-2-ol

Followed the coupling procedure described for Step 1 of Example 7,starting from 6-bromonaphthalen-2-ol and1H-benzo[d][1,2,3]triazol-6-ylboronic acid where the base used was K₂CO₃and the reaction was run in the microwave for 2 h at 150° C.Purification by flash chromatography with a mobile phase gradient of 0%to 75% EtOAc in hexanes gave Example 19 as an off-white powder (7.5 mg,6.4% yield). ¹H NMR (DMSO-d₆ 500 MHz): δ 9.84 (s, 1H), 8.20 (s, 2H),8.03 (d, 1H), 7.90 (m, 4H), 7.17 (m, 2H); MS (ESI): m/z 262.06 [M+1]⁺.

Example 20 4-(6-hydroxynaphthalen-2-yl)-3-(trifluoromethyl)benzoic acid

Step 1: Synthesis of4-(6-methoxynaphthalen-2-yl)-3-(trifluoromethyl)benzoic acid: To amixture of methyl 4-bromo-3-(trifluoromethyl)benzoate (Intermediate 5)(5.20 g), 2-bromo-6-methoxy-naphthalene (1.50 g, 6.33 mmol), and K₂CO₃(1.75 g, 12.7 mmol) in DEGME/H₂O (70 mL/10 mL) was added Pd(dppf)Cl₂(114 mg, 0.139 mmol) under N₂ atmosphere. The mixture was heated to 120°C. for 3 hours. H₂O (150 mL) was added, extracted with EtOAc (50 mL×2).The aqueous layer was acidified with 1N aqueous HCl to pH=3-4, extractedwith EtOAc (50 mL×3), washed with brine (50 mL), dried over anhydrousNa₂SO₄, filtered and concentrated. The crude product was purified bysilica gel column (PE/EtOAc=10/1 to 1/1) to give the product (1.10 g) asa brown solid.

Step 2: Synthesis of Example 20: To a solution of the above product(1.10 g, from above crude) in anhydrous DCM (15 mL) was added BBr₃ (2.0mL, 21.1 mmol, d=2.64 g/mL) dropwise at 0° C. The mixture was stirred at25° C. for 16 hours, then quenched with H₂O (90 mL) and extracted withDCM (30 mL×3). The combined organic layers were washed with brine (30mL), dried over anhydrous Na₂SO₄, filtered and concentrated to give thecrude product which was purified by prep-HPLC (0.1% TFA as additive).Most of CH₃CN was removed under reduced pressure and the remainingsolvent was removed by lyophilization to give Example 20 (21 mg) as anoff-white solid. ¹H NMR (MeOD 400 MHz): δ 8.40 (s, 1H), 8.27 (d, J=8.0Hz, 1H), 7.74 (d, J=8.8 Hz, 1H), 7.72-7.68 (m, 2H), 7.56 (d, J=8.0 Hz,1H), 7.34 (d, J=8.8 Hz, 1H), 7.19-7.08 (m, 2H). MS (ESI): m/z 331.0[M−H]⁻.

Example 21 3-chloro-4-(3-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of3-chloro-4-(3-fluoro-6-methoxynaphthalen-2-yl)benzoic acid: Followed thecoupling procedure described in Step 1 of Example 20, where the startingmaterials were 3-fluoro-2-iodo-6-methoxynaphthalene (Intermediate 6) and4-carboxy-2-chlorophenylboronic acid and wherein the reaction was heatedto 90° C. for 3 hours. The desired product (380 mg, yield 70%) wasisolated as a solid after workup.

Step 2: Synthesis of Example 21: Followed the BBr₃ deprotectiondescribed for Example 20, Step 2. Example 21 was isolated (45 mg, yield12%) as an off-white solid. ¹H NMR (DMSO-d₆ 400 MHz): δ 13.41 (brs, 1H),10.05 (brs, 1H), 8.06 (d, J=1.6 Hz, 1H), 7.99 (dd, J=8.0, 1.6 Hz, 1H),7.85 (d, J=8.0 Hz, 2H), 7.68-7.56 (m, 2H), 7.17 (d, J=2.4 Hz, 1H), 7.10(dd, J=8.8, 2.4 Hz, 1H). MS (ESI): m/z 315.0 [M−H]⁻.

Example 22 4-(3-chloro-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of 4-(3-chloro-6-methoxynaphthalen-2-yl)benzoic acid:Followed the coupling procedure described in Step 1 of Example 20,starting from 3-chloro-2-iodo-6-methoxynaphthalene (Intermediate 7) and4-carboxyphenylboronic acid, where the reaction was stirred under N₂atmosphere at 90° C. for 4 hours. After workup, the residue wastriturated with DCM (50 mL) to give the product (220 mg, yield: 75%) asan off-white solid.

Step 2: Synthesis of Example 22: A mixture of the above product (100 mg,0.320 mmol) and BBr₃ (0.4 mL, 4.2 mmol) in anhydrous DCM (2.5 mL) wasstirred at 15° C. for 2 days. Followed the workup/purification proceduredescribed in Step 2 of Example 20 to give Example 22 (24 mg, yield 25%)as off-white solid. ¹H NMR (DMSO-d₆ 400 MHz): δ 13.02 (brs, 1H), 10.04(brs, 1H), 8.02 (d, J=8.0 Hz, 2H), 7.96 (s, 1H), 7.87 (s, 1H), 7.84 (d,J=8.8 Hz, 1H), 7.61 (d, J=8.0 Hz, 2H), 7.16-7.08 (m, 2H). MS (ESI): m/z297.0 [M−H]⁻.

Example 23 4-(3-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of 4-(3-fluoro-6-methoxynaphthalen-2-yl)benzoic acid:Followed the coupling procedure described in Step 1 of Example 20,starting from 3-fluoro-2-iodo-6-methoxynaphthalene (Intermediate 6) and4-carboxyphenylboronic acid. The reaction was heated to 90° C. for 3hours. The product was isolated after workup (80 mg, crude).

Step 2: Synthesis of Example 23: Followed the BBr₃ deprotectiondescribed for Example 20, Step 2. Example 23 (11 mg, 2-step yield 2%)was isolated as an off-white solid. ¹H NMR (MeOD 400 MHz): δ 8.10 (d,J=8.4 Hz, 2H), 7.89 (d, J=8.0 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 7.71 (d,J=6.8 Hz, 2H), 7.40 (d, J=12.4 Hz, 1H), 7.09 (d, J=2.0 Hz, 1H), 7.05(dd, J=8.8, 2.0 Hz, 1H). MS (ESI): m/z 281.0 [M−H]⁻.

Example 24 4-(6-hydroxy-1-methoxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of 4-(6-(benzyloxy)-1-methoxynaphthalen-2-yl)benzoicacid: Followed the coupling procedure described in Step 1 of Example 20,where the starting materials were6-(benzyloxy)-2-bromo-1-methoxynaphthalene (Intermediate 9) and4-carboxyphenylboronic acid and where the reaction was stirred at 130°C. for 5 hours. The product (96 mg, yield 43%) was isolated as a yellowsolid.

Step 2: Synthesis of Example 24: A mixture of the above product (96 mg,0.25 mmol) and 10% Pd/C (100 mg, 50% wet) in EtOAc (10 mL) was stirredunder H₂ (15 psi) at 30° C. for 18 hours. The mixture was filtered andthe filtrate was concentrated in vacuo. The residue was purified byprep-TLC (PE/EtOAc=2/1) to give Example 24 (13.5 mg, yield 19%) as anoff-white solid. ¹H NMR (CD₃OD 400 MHz TMS): δ 8.18-8.06 (m, 3H), 7.78(d, J=8.4 Hz, 2H), 7.51 (d, J=8.8 Hz, 1H), 7.42 (d, J=8.4 Hz, 1H), 7.14(s, 1H), 7.13 (d, J=7.6 Hz, 1H), 3.55 (s, 3H). MS (ESI): m/z 293.0[M−H]⁻.

Example 25 4-(1-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl4-(1-fluoro-6-methoxynaphthalen-2-yl)benzoate: A mixture of1-fluoro-6-methoxynaphthalen-2-yltrifluoromethanesulfonate (Intermediate10) (80.0 mg, 0.247 mmol), 4-methoxycarbonylphenylboronic acid (44.5 mg,0.247 mmol) and aqueous Na₂CO₃ (2M, 0.27 mL, 0.54 mmol) in toluene/EtOH(4 mL/1 mL) was degassed three times under N₂ atmosphere. Then Pd(PPh₃)₄(28.6 mg, 0.0247 mmol) was added and the mixture was stirred at 80° C.for 5 hours under N₂ atmosphere. An aqueous/EtOAc workup was followed bysilica gel column chromatography (PE/EtOAc=200/1 to 100/1) to give theproduct (50 mg, yield 65%) as an off-white solid.

Step 2: Synthesis of methyl4-(1-fluoro-6-hydroxynaphthalen-2-yl)benzoate: To a solution of theabove product (50.0 mg, 0.161 mmol) in anhydrous DCM (5 mL) was addedBBr₃ (0.20 mL, 2.1 mmol, d=2.64 g/mL) dropwise at 0° C. The resultingmixture was stirred at 25° C. for 16 hours. An aqueous/DCM workup gavethe product (50 mg, crude) as an off-white solid, which was used fornext step directly. MS (ESI): m/z 295.0 [M−H]⁻.

Step 3: Synthesis of Example 25: Followed the hydrolysis proceduredescribed for Step 3 of Example 13 to give Example 25 (5 mg, 2-stepyield: 11%) as a white solid. ¹H NMR (MeOD 400 MHz): δ 8.12 (d, J=8.4Hz, 2H), 8.02 (d, J=8.8 Hz, 1H), 7.75 (d, J=7.6 Hz, 2H), 7.58-7.47 (m,2H), 7.21-7.12 (m, 2H). MS (ESI): m/z 281.0 [M−H]⁻.

Example 26 4-(6-hydroxy-3-methylnaphthalen-2-yl)benzoic acid

Step 1: Synthesis of4-(6-(benzyloxy)-3-(methoxymethoxy)naphthalen-2-yl)benzoic acid:Followed the coupling procedure described in Step 1 of Example 20, wherethe starting materials were6-(benzyloxy)-2-bromo-3-(methoxymethoxy)naphthalene (Intermediate 11)and 4-methoxycarbonylphenylboronic acid and where the reaction wasstirred at 90° C. for 3 hours. The desired product (1.30 g, yield 72%)was obtained as an off-white solid.

Step 2: Synthesis of methyl4-(6-(benzyloxy)-3-(methoxymethoxy)naphthalen-2-yl)benzoate: A mixtureof the above product (1.20 g, 2.90 mmol), K₂CO₃ (800 mg, 5.80 mmol) andCH₃I (824 mg, 5.80 mmol) in DMF (15 mL) was stirred at 10° C. for 4hours. The mixture was neutralized with aqueous HCl (0.1M) until pH=7and extracted with EtOAc (50 mL×3). The combined organic layers werewashed with brine (100 mL), dried over Na₂SO₄ and concentrated in vacuoto give the product (1.10 g, yield 89%) as an off-white solid.

Step 3: Synthesis of methyl4-(6-(benzyloxy)-3-hydroxynaphthalen-2-yl)benzoate: To a solution of theabove product (1.10 g, 2.57 mmol) in THF/MeOH (8 mL/2 mL) was addedconc. HCl (0.2 mL). The mixture was refluxed for 3 hours. The mixturewas concentrated under reduced pressure to give the crude product, whichwas washed with PE (50 mL) and EtOAc (30 mL) to give the product (900mg, yield 91%) as white solid.

Step 4: Synthesis of methyl4-(6-(benzyloxy)-3-(((trifluoromethyl)sulfonyl)oxy)naphthalen-2-yl)benzoate:To a solution of the above product (615 mg, 1.60 mmol) and Et₃N (1.30 g,12.9 mmol) in DCM (20 mL) was added Tf₂O (903 mg, 3.20 mmol) dropwiseand then the mixture was stirred at 10° C. for 30 minutes. After anaqueous/EtOAc workup, the residue was purified by silica gel column(PE/EtOAc=40/1) to give the product (450 mg, yield: 54%) as an off-whitesolid.

Step 5: Synthesis of methyl4-(6-(benzyloxy)-3-methylnaphthalen-2-yl)benzoate: To a solution ofZnCl₂ (1.32 g, 9.68 mmol) in anhydrous THF (20 mL) was added MeMgCl (1.6mL, 4.80 mmol, 3M in THF) and the mixture was stirred at under N₂atmosphere at 10° C. for 1 hour. Then methyl4-(6-(benzyloxy)-3-(((trifluoromethyl)sulfonyl)oxy)naphthalen-2-yl)benzoate(500 mg, 0.968 mmol) and Pd(PPh₃)₄ (100 mg, 0.0865 mmol) was added andthe mixture was stirred at 60° C. under N₂ atmosphere for 3 hours. Afteran aqueous/EtOAc workup, the residue was purified by silica gel column(PE/EtOAc=40/1) to give the product (250 mg, yield 68%) as an off-whitesolid.

Step 6: Synthesis of 4-(6-(benzyloxy)-3-methylnaphthalen-2-yl)benzoicacid: A mixture of the above product (150 mg, 0.392 mmol) and aqueousNaOH (10 mL, 2M) in MeOH (10 mL) was refluxed overnight. The mixture wascooled to room temperature, acidified with aqueous HCl (2M) until pH=5and extracted with EtOAc (50 mL×3), dried over Na₂SO₄ and concentratedin vacuo to give the product (100 mg, yield 69%) as off-white solid.

Step 7: Synthesis of Example 26: A mixture of the above product (100 mg,0.271 mmol) and 10% Pd/C (50 mg, 50% wet) in EtOAc (10 mL) was stirredat 20° C. for 2 days. The mixture was filtered and the filtrate wasconcentrated under reduced pressure. The residue was purified byprep-HPLC (0.1% TFA as additive) to give Example 26 (60 mg, yield 79%)as off-white solid. ¹H NMR (CD₃OD 400 MHz TMS): δ 8.09 (d, J=8.0 Hz,2H), 7.69 (d, J=8.8 Hz, 1H), 7.58 (s, 1H), 7.54 (s, 1H), 7.49 (d, J=8.0Hz, 2H), 7.06 (d, J=2.4 Hz, 1H), 7.02 (dd, J=8.4, 2.4 Hz, 1H), 2.35 (s,3H). MS (ESI): m/z 277.0 [M−H]⁻.

Example 27 4-(1-cyano-5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

Step 1: Synthesis of methyl4-(1-cyano-5-fluoro-6-methoxynaphthalen-2-yl)benzoate: To a solution ofmethyl 4-(1-cyano-6-methoxynaphthalen-2-yl)benzoate (1.66 g, 5.23 mmol)(described in step 1 of Example 15) in CH₃CN (30 mL) was addedSelectfluor (2.04 g, 5.75 mmol). The reaction mixture was heated at 60°C. for 3 h and concentrated. The residue was partitioned between water(20 mL) and EtOAc (50 mL). The aqueous phase was separated and extractedwith EtOAc. The combined organic layers were washed with brine, driedover MgSO₄, filtered and concentrated. The residue was purified bysilica gel chromatography (PE/EA=4/1) to give the product (1.1 g,crude).

Step 2: Synthesis of Example 27: To an ice cooled solution of the aboveproduct (1.1 g, 3.3 mmol) in DCM (2 mL) was added BBr₃ (3 M in DCM, 10mL, 30 mmol). The reaction mixture was stirred at room temperatureovernight and slowly quenched with water and extracted with EtOAc. Thecombined organic layers were dried over MgSO₄, filtered andconcentrated. The residue was washed with a mixture of DMF and CH₃CN.The solid was washed by CH₃CN, dried in high vacuum to give Example 27(0.31 g, 31%) as white powder. ¹H-NMR (DMSO-d₆, 500 MHz, TMS): δ 13.19(s, 1H), 10.75 (s, 1H), 8.31 (d, J=9.0 Hz, 1H), 8.12 (d, J=8.0 Hz, 2H),7.93 (d, J=9.0 Hz, 1H), 7.83 (d, J=8.0 Hz, 2H), 7.78 (d, J=9.0 Hz, 1H),7.60 (t, J=8.5 Hz, 1H). MS (ESI): m/z=306.0 [M−1]⁺.

Example 28 4-(1-cyano-6-hydroxynaphthalen-2-yl)-3-fluorobenzoic acid

Step 1: Synthesis of methyl4-(1-cyano-6-methoxynaphthalen-2-yl)-3-fluorobenzoate: Followedprocedure described for Example 6, step 1 with modification: Pd(PPh₃)₄was the catalyst, sodium carbonate was the base, and solvent used wastoluene/EtOH/water (5/2/1). Purification by silica gel chromatography(PE/EA=2/1) gave the desired product (430 mg, 85%) as white solid.

Step 2: Synthesis of Example 28: To an ice cooled solution of the aboveproduct (280 mg, 0.83 mmol) in DCM (2 mL) was added BBr₃ (3 M in DCM, 3mL, 9 mmol). The reaction mixture was stirred at room temperatureovernight and slowly quenched with water (40 mL). The precipitate wascollected by filtration and purified by prep-HPLC to give Example 28(132 mg, 51%) as yellow solid. ¹H-NMR (DMSO-d₆, 500 MHz, TMS): δ 13.48(s, 1H), 10.41 (s, 1H), 8.20 (d, J=8.5 Hz, 1H), 8.07 (d, J=8.5 Hz, 1H),7.94 (dd, J=9.0 Hz, 1H), 7.87 (dd, J=11.0 Hz, 1H), 7.76 (t, J=7.5 Hz,1H), 7.61 (d, J=8.5 Hz, 1H), 7.43 (dd, J=9.5 Hz, 1H), 7.39 (d, J=2.0 Hz,1H). MS (ESI): m/z=308.1 [M+1]⁺.

Example 29 3-chloro-4-(5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

6-Bromo-1-fluoronaphthalen-2-ol (Intermediate 13, 1.0 mmol) and4-borono-3-chlorobenzoic acid (2.0 mmol) were mixed with 5% (Ph₃P)₄Pdand NaHCO₃ (4.0 mmol) in 20 ml of 50% dioxane/water. The mixture wasdegassed three times by evacuation and argon filling and heated at 95°C. overnight. The reaction was diluted with 20 ml of water and filtered.The filtrate was acidified with 1N HCl to pH=4. The precipitate wasfiltered and washed with water, dried. The crude was purified by columnusing AcOH/MeOH/EtOAC (1/5/94) as solvent B and hexanes as solvent Awith gradient 2 to100% B to give a white solid. The solid was trituratedwith diisopropyl ether to give Example 29 (50 mg). 1H-NMR (DMSO-d₆, 300MHz, TMS): δ 13.40 (b, 1H), 10.25 (b, 1H), 8.06 (s, 1H), 7.91-7.97 (m,3H), 7.62-7.71 (m, 3H), 7.32 (t, 1H), MS (ESI): m/z=315.3 [M−1]⁻.

Example 30 4-(5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

Followed the coupling procedure described in Example 29 starting from6-bromo-1-fluoronaphthalen-2-ol (Intermediate 13) and 4-boronobenzoicacid. ¹H-NMR (DMSO-d₆, 300 MHz, TMS): δ 13.04 (b, 1H), 10.23 (b, 1H),8.29 (s, 1H), 7.91-8.01 (m, 7H), 7.74 (d, 1H). MS (ESI): m/z=281.26[M−1]⁻.

Example 31 3-fluoro-4-(5-fluoro-6-hydroxynaphthalen-2-yl)benzoic acid

Followed the coupling procedure described in Example 29 starting from6-bromo-1-fluoronaphthalen-2-ol (Intermediate 13) and4-borono-3-fluorobenzoic acid. ¹H-NMR (DMSO-d₆, 300 MHz, TMS): δ 13.32(b, 1H), 10.28 (b, 1H), 8.14 (s, 1H), 7.98 (d, 1H), 7.78 (d, 1H),7.73-7.76 (m, 4H), 7.32 (t, 1H), MS (ESI): m/z=299.40 [M−1]⁻.

Example 32 4-(5-fluoro-6-hydroxynaphthalen-2-yl)-3-methylbenzoic acid

Followed the coupling procedure described in Example 29 starting from6-bromo-1-fluoronaphthalen-2-ol (Intermediate 13) and methyl3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate. ¹H-NMR(DMSO-d₆, 300 MHz, TMS): δ 12.90 (b, 1H), 10.17 (b, 1H), 7.84-7.97 (m,4H), 7.70 (d, 1H), 7.54 (d, 1H), 7.41 (d, 1H), 7.30 (t, 1H), 2.33 (s,3H). MS (ESI): m/z=295.25 [M−1]⁻.

Example 33 4-(6-hydroxynaphthalen-2-yl)benzoic acid

Followed the coupling procedure described in Example 3, starting from6-bromonaphthalen-2-ol and 4-boronobenzoic acid, where the reaction washeated at 85° C. for 8 h. After the prep-HPLC purification, a furtherpurification by silica gel column (PE:EA=7:1 to 4:1) gave the compoundas a yellow solid (26 mg, 7%). ¹H NMR (DMSO-d6 500 MHz TMS): δ 12.97(brs, 1H), 9.91 (brs, 1H), 8.19 (s, 1H), 8.04 (d, J=8.0 Hz, 2H),7.87˜7.92 (m, 3H), 7.80 (m, 2H), 7.13˜7.16 (m, 2H); MS (ESI): m/z 265.1[M+1]⁺.

Example 34 Synthesis of Intermediates Intermediate 1: methyl3-cyano-4-(trifluoromethylsulfonyloxy)benzoate

Step 1: Synthesis of methyl 3-cyano-4-hydroxybenzoate: A mixture ofmethyl 3-bromo-4-hydroxybenzoate (2.50 g, 10.8 mmol) and CuCN (1.10 g,12.3 mmol) in NMP (10 mL) was stirred at 200° C. under N₂ atmosphere for3 hours. The reaction mixture was cooled to room temperature andfiltered. The filtrate was diluted in ethyl acetate (100 mL) and washedwith water (50 mL×3) and brine (50 mL), then dried over anhydrous Na₂SO₄and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (PE/EtOAc=8/1).

Step 2: Synthesis of Intermediate 1: To a mixture of crude methyl3-cyano-4-hydroxybenzoate (2.50 g) from above and Et₃N (1.31 g, 13.0mmol) in anhydrous DCM (20 mL) was added Tf₂O (1.82 g, 6.50 mmol)dropwise and then the reaction mixture was stirred at 30° C. for 2hours. The resulting mixture was suspended in water (50 mL) andextracted with DCM (50 mL×3), dried over anhydrous Na₂SO₄ andconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (PE/EtOAc=10/1) to give Intermediate 1 (550 mg, 2-stepyield: 28%) as a colorless oil. ¹H NMR (CDCl₃ 300 MHz): δ 8.43 (d, J=2.1Hz, 1H), 8.37 (dd, J=8.7, 2.1 Hz, 1H), 7.59 (d, J=8.7 Hz, 1H), 3.98 (s,3H).

Intermediate 2: 1-bromo-6-methoxynaphthalen-2-yltrifluoromethanesulfonate

Step 1: Synthesis of 1-bromo-6-methoxynaphthalen-2-ol: To a solution of6-methoxynaphthalen-2-ol (2.00 g, 11.5 mmol) in DMF (20 mL) was addedNBS (2.15 g, 12.1 mmol) and the reaction mixture was stirred at 25° C.for 4 hours. The resulting mixture was diluted with EtOAc (300 mL),washed with H₂O (100 mL×5) and brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated in vacuo to give the crude product, which waspurified by silica gel column (PE/EtOAc=20/1) to give the product (2.10g, yield 72%).

Step 2: Synthesis of 1-bromo-6-methoxynaphthalen-2-yltrifluoromethanesulfonate: To a solution of above product (2.70 g, 10.7mmol) and TEA (1.41 g, 13.9 mmol) in anhydrous DCM (30 mL) was addedTf₂O (3.33 g, 11.8 mmol) at −50° C. and the reaction mixture was stirredat the same temperature for 0.5 hour. The resulting mixture was quenchedwith brine (150 mL), extracted with DCM (50 mL×3). The combined organiclayers were washed with brine (50 mL), dried over anhydrous Na₂SO₄ andconcentrated in vacuo to give crude Intermediate 2 (3.80 g) as a solid.¹H NMR (CDCl₃ 400 MHz): δ 8.17 (d, J=9.6 Hz, 1H), 7.74 (d, J=9.2 Hz,1H), 7.36 (d, J=9.2 Hz, 1H), 7.31 (dd, J=9.2, 2.4 Hz, 1H), 7.14 (d,J=2.8 Hz, 1H), 3.93 (s, 3H).

Intermediate 3: 6-bromo-7-methoxynaphthalen-2-ol

Step 1: Synthesis of 3-bromonaphthalene-2,7-diol: To a solution ofnaphthalene-2,7-diol (5.00 g, 31.3 mmol) in AcOH (25 mL) was added Br₂(3.3 mL, 62.6 mmol) in AcOH (25 mL) dropwise over 15 minutes at 10-15°C., then mixture was stirred at 10-15° C. for 1 hour. Sn powder (7.75 g,64.6 mmol) and H₂O (20 mL) was added and the mixture was heated to 80°C. for 1 hour. The mixture was diluted with ice-water (50 mL), theextracted with EtOAc (30 mL×3), organic was washed with brine (50 mL),dried over Na₂SO₄, filtered and concentrated. Purification by columnchromatography on silica gel (PE/EtOAc=15/1) and further prep-HPLC (0.1%TFA as additive) gave the product (2.0 g, yield 27%) as a solid.

Step 2: Synthesis of 6-bromo-7-methoxynaphthalen-2-ol: To a solution ofabove product (500 mg, 2.08 mmol) in DMF (12 mL) was added K₂CO₃ (579mg, 4.17 mmol). The mixture was stirred at 25° C. for 20 minutes underN₂. A solution of CH₃I (260 mg, 1.83 mmol) in DMF (1 mL) was added viasyringe pump over 2 hours. The mixture was stirred at 25° C. for 30minutes. The mixture was acidified with 2M HCl to pH=5. The mixture wasextracted with DCM (20 mL×3). The combined organic layers were washedwith brine (50 mL), dried over anhydrous Na₂SO₄ and concentrated invacuo, then purified by column chromatography on silica gel(PE/EtOAc=10/1) to give Intermediate 3 (260 mg, yield 49%) as a whitesolid. ¹H NMR (CDCl₃ 400 MHz): δ 7.96 (s, 1H), 7.59 (d, J=8.4 Hz, 1H),7.05 (d, J=2.4 Hz, 1H), 7.00 (s, 1H), 6.97 (dd, J=8.8, 2.4 Hz, 1H), 5.05(s, 1H), 3.91 (s, 3H).

Intermediate 4: 1-chloro-6-methoxynaphthalen-2-yltrifluoromethanesulfonate

Step 1: Synthesis of 1-chloro-6-methoxynaphthalen-2-ol: To a solution of6-methoxynaphthalen-2-ol (1.00 g, 5.74 mmol) in DMF (15 mL) was addedNCS (843 mg, 6.31 mmol). The reaction mixture was stirred at 25° C. for16 hours. Diluted the reaction with EtOAc, washed with H₂O and brine,dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo.Purification by silica gel column (PE/EtOAc=100/1) gave the product (940mg, yield 87%).

Step 2: Synthesis of 1-chloro-6-methoxynaphthalen-2-yltrifluoromethanesulfonate: Followed procedure described in Step 2 ofIntermediate 2. ¹H NMR (CDCl₃ 400 MHz): δ 8.21 (d, J=9.2 Hz, 1H), 7.71(d, J=9.2 Hz, 1H), 7.39 (d, J=9.2 Hz, 1H), 7.34 (dd, J=9.2, 2.4 Hz, 1H),7.16 (d, J=2.4 Hz, 1H), 3.95 (s, 3H).

Intermediate 5: methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)benzoate

To a mixture of methyl 4-bromo-3-(trifluoromethyl)benzoate (2.00 g, 7.07mmol), bis(pinacolato)diboron (3.60 g, 14.1 mmol), and KOAc (2.08 g,21.2 mmol) in DMSO (30 mL) was added Pd(PPh₃)₄ (1.63 g, 1.41 mmol) underN₂ atmosphere. Then the mixture was heated to 120° C. for 3 hours. Thereaction mixture was diluted with EtOAc (150 mL). The organic phase wasseparated, washed with H₂O (50 mL×3) and brine (50 mL), dried overanhydrous Na₂SO₄, filtered and concentrated to give the crude product(5.2 g) as a yellow oil.

Intermediate 6: 3-fluoro-2-iodo-6-methoxynaphthalene

Step 1: Synthesis of tert-butyl (7-methoxynaphthalen-2-yl)carbamate: Amixture of 7-methoxynaphthalen-2-amine (84.0 g, 485 mmol) and Boc₂O (116g, 534 mmol) in THF (500 mL) was stirred at 65° C. overnight. Themixture was concentrated. Purification by silica gel column(PE/EtOAc=50/1) gave the product (95.0 g, yield 71%) as off-white solid.

Step 2: To a mixture of the above product (20.0 g, 73.2 mmol) inanhydrous THF (1000 mL) was added t-BuLi (350 mL, 455 mmol, 1.3 M inpentane) dropwise at −20° C. under N₂ atmosphere. The reaction mixturewas stirred at −10° C. for 30 minutes. Then 1,2-diiodoethane (51.6 g,183 mmol) was added and the mixture was stirred at 20° C. for 1 hour.The mixture was quenched with water (1000 mL) and extracted with ethylacetate (1000 mL×3). The combined organic layers were dried overanhydrous Na₂SO₄, concentrated, and then purified by silica gel column(PE/EtOAc=200/1˜5/1) to give tert-butyl(6-iodo-7-methoxynaphthalen-2-yl)carbamate (8.15 g, yield 28%) and amixture of isomers (8.55 g) as off-white solid.

Step 3: Synthesis of 3-iodo-7-methoxynaphthalen-2-amine: A mixture oftert-butyl (6-iodo-7-methoxynaphthalen-2-yl)carbamate with other isomer(8.00 g from above) and TFA (30 mL) in DCM (90 mL) was stirred at 20° C.for 3 hours, then concentrated. Aqueous saturated NaHCO₃ (200 mL) wasadded, then extracted with ethyl acetate (200 mL×3). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated.Purification by silica gel column (PE/EtOAc=5/1) gave the product (3.50g, 2-step yield 16%) as off-white solid.

Step 4: Synthesis of Intermediate 6: To a solution of the above product(1.50 g, 5.01 mmol) in water (20 mL) and conc. HCl (20 mL) was added asolution of NaNO₂ (345 mg, 5.01 mmol) in water (10 mL) dropwise at 0° C.The reaction was stirred at 0° C. for 1 hour. Then HBF₄ (10 mL) wasadded and the mixture was stirred for 10 minutes. The mixture wasfiltered and the solid was washed with water (50 mL), dried underreduced pressure. The solid was dissolved in xylene (20 mL) and refluxedfor 1 hour. The mixture was cooled to room temperature, diluted withwater (50 mL), extracted with ethyl acetate (50 mL×3). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated invacuo. The residue was purified by silica gel column (PE/EtOAc=100/1) togive the product (1.20 g, 79%). ¹H NMR (CDCl₃ 400 MHz TMS): δ 8.19 (d,J=6.0 Hz, 1H), 7.63 (d, J=9.2 Hz, 1H), 7.38 (d, J=9.2 Hz, 1H), 7.10 (dd,J=9.2, 2.4 Hz, 1H), 7.03 (d, J=2.4 Hz, 1H), 3.92 (s, 3H).

Intermediate 7, 3-chloro-2-iodo-6-methoxynaphthalene

To a solution of 3-iodo-7-methoxynaphthalen-2-amine (see Intermediate 6,product of step 3) (1.0 g, 3.34 mmol) in water (20 mL) and conc. HCl (20mL) was added a solution of NaNO₂ (230 mg, 3.34 mmol) in water (10 mL)dropwise at 0° C. and the reaction was stirred at 0° C. for 1 hour. ThenCuCl (400 mg, 4.04 mmol) was added and the mixture was stirred at 20° C.for 2 hours. An aqueous/EtOAc workup was followed by purification bysilica gel column (PE/EtOAc=100/1) to give3-chloro-2-iodo-6-methoxynaphthalene (900 mg, yield 85%) as off-whitesolid. ¹H NMR (CDCl₃ 300 MHz TMS): δ 8.29 (s, 1H), 7.84 (s, 1H), 7.60(d, J=9.0 Hz, 1H), 7.13 (dd, J=9.0, 2.7 Hz, 1H), 6.99 (d, J=2.4 Hz, 1H),3.91 (s, 3H).

Intermediate 8, MPHT

To MeOH (200 mL) was added HBr (97.2 g, 1.20 mol) in HOAc (120 mL)dropwise followed by the addition of Br₂ (190 g, 1.20 mol). The mixturewas stirred at 10° C. for 10 minutes. Then NMP (257 g, 2.60 mol) wasadded dropwise. The reaction mixture was stirred at 10° C. for 1 hour.Then the mixture was filtered. The solid was washed with MTBE (200 mL)and dried under vacuum to give MPHT (361 g, yield 41%) as an orangesolid. ¹H NMR (CDCl₃ 400 MHz): δ 3.72 (t, J=7.2 Hz, 4H), 3.07 (s, 6H),2.92 (t, J=8.0 Hz, 4H), 2.32-2.18 (m, 4H).

Intermediate 9, 6-(benzyloxy)-2-bromo-1-methoxynaphthalene

Step 1: Synthesis of 6-(benzyloxy)-3,4-dihydronaphthalen-1(2H)-one: Amixture of 6-hydroxy-1-tetralone (50.0 g, 308 mmol), K₂CO₃ (64.0 g, 434mmol) and BnBr (58.0 g, 340 mmol) in DMF (400 mL) was stirred at 25° C.for 16 hours. The mixture was diluted in water (1000 mL), extracted withEtOAc (1000 mL×3). The combined organic layers were washed with brine(500 mL×3), dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to give the product as a brown solid (54.0 g, yield 69%).

Step 2: Synthesis of6-(benzyloxy)-2,2-dibromo-3,4-dihydronaphthalen-1(2H)-one: To a mixtureof the above product (10.1 g, 40.0 mmol) in MeCN (20 mL) was added asolution of MPHT (Intermediate 8) (35.1 g, 80.0 mmol) in MeCN (130 mL)dropwise at 80° C. Then the reaction mixture was stirred at 80° C. for1.5 hours. The mixture was quenched with aqueous saturated Na₂S₂O₃ (200mL) and extracted with EtOAc (300 mL×3). The combined organic layerswere washed with 5% aqueous HCl (100 mL×3), dried over anhydrous Na₂SO₄and concentrated in vacuo. The residue was purified by silica gel column(PE/EtOAc=50/1) to give the product (7.18 g, yield 44%) as a whitesolid.

Step 3: Synthesis of 6-(benzyloxy)-2-bromonaphthalen-1-ol: A mixture ofthe above product (7.18 g, 17.5 mmol) and TEA (50 mL) in anhydrous CHCl₃(30 mL) was stirred at 25° C. for 4 hours. The mixture was quenched withaqueous saturated Na₂S₂O₃ (200 mL) and extracted with DCM (300 mL×3).The combined organic layers were washed with 5% aqueous HCl (100 mL×3),dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue waspurified by silica gel column (PE) to give the product (450 mg, yield8%) as a white solid.

Step 4: Synthesis of Intermediate 9: A mixture of the above product (500mg, 1.51 mmol), K₂CO₃ (415 mg, 3.00 mmol) and CH₃I (0.75 mL, 14.8 mmol,2.80 g/mL) in DMF (10 mL) under N₂ atmosphere was stirred at 25° C. for18 hours. The mixture was diluted in water (50 mL) and extracted withEtOAc (50 mL×3), dried over anhydrous Na₂SO₄ and concentrated. Theresidue was purified by prep-TLC (PE/EtOAc=100/1) to give6-(benzyloxy)-2-bromo-1-methoxynaphthalene (321 mg, yield 59%) as anoff-white solid. ¹H NMR (CDCl₃ 300 MHz TMS): δ 8.04 (d, J=9.0 Hz, 1H),7.56-7.46 (m, 3H), 7.46-7.34 (m, 4H), 7.30-7.24 (m, 1H), 7.19 (d, J=2.4Hz, 1H), 5.17 (s, 2H), 3.99 (s, 3H).

Intermediate 10, 1-fluoro-6-methoxynaphthalen-2-yltrifluoromethanesulfonate

Step 1: Synthesis of 1-bromo-6-methoxy-2-(methoxymethoxy)naphthalene: Toa solution of 1-bromo-6-methoxynaphthalen-2-ol (described in U.S.61/423,799) (2.90 g, 11.5 mmol) in MeOH (20 mL) and THF (20 mL) wasadded K₂CO₃ (3.18 g, 23.0 mmol) and MOMCl (1.10 g, 13.8 mmol). Theresulting mixture was stirred at 20° C. for 48 hours and stirred at30-40° C. for 5 days. An aqueous/EtOAc workup was followed bypurification by silica gel column chromatography (PE to PE/EtOAc=200/1)to give the product (1.90 g, yield 56%) as an off-white solid.

Step 2: Synthesis of 1-fluoro-6-methoxy-2-(methoxymethoxy)naphthalene:To a solution of the above product (1.00 g, 3.38 mmol) in anhydrous THF(20 mL) was added n-BuLi (1.62 mL, 4.06 mmol, 2.50 M in hexane) dropwiseat 0° C. The mixture was stirred at 0° C. for 30 min then cooled to −78°C. A solution of N-fluorobenzenesulfonimide (1.28 g, 4.06 mmol) inanhydrous THF (5 mL) was added to the reaction mixture. The resultingmixture was stirred at −78° C. for 1 hour, then stirred at 25° C. for 12hours. The reaction mixture was quenched with aq. NH₄Cl (60 mL), andextracted with EtOAc (20 mL×2). The combined organic layers were washedwith H₂O (20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. The crude product waspurified by silica gel column (PE to PE/EtOAc=200/1) to give the product(280 mg, yield 35%) as an off-white solid.

Step 3: Synthesis of 1-fluoro-6-methoxynaphthalen-2-ol: A mixture of theabove product (770 mg, 3.26 mmol) in HCl/dioxane (15 mL) was stirred at25° C. for 2 hours. The reaction mixture was neutralized with aqueousNaOH (2M) to pH=7, followed by an aqueous/EtOAc workup. The crudeproduct was purified by silica gel column (PE/EtOAc=200/1 to 100/1) togive the product (380 mg, yield 61%) as an off-white solid.

Step 4: Synthesis of Intermediate 10: To a solution of the above product(50.0 mg, 0.260 mmol) and TEA (34.2 mg, 0.338 mmol) in anhydrous DCM (10mL) was added Tf₂O (80.7 mg, 0.286 mmol) at −50° C. The reaction mixturewas stirred at −50° C. for 0.5 hour. The resulting mixture was quenchedwith brine (30 mL) and extracted with DCM (20 mL×3). The combinedorganic layers were washed with H₂O (20 mL) and brine (20 mL), driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto give Intermediate 10 (80 mg, yield 95%) as an off-white solid. ¹H NMR(CDCl₃ 400 MHz): δ 8.04 (d, J=9.2 Hz, 1H), 7.55 (d, J=8.8 Hz, 1H),7.38-7.21 (m, 2H), 7.15 (s, 1H), 3.94 (s, 3H).

Intermediate 11, 6-(benzyloxy)-2-bromo-3-(methoxymethoxy)naphthalene

Step 1: Synthesis of 3-bromonaphthalene-2,7-diol: To a solution of2,7-dihydroxynaphthalene (8.00 g, 50.0 mmol) in AcOH (30 mL) was addedBr₂ (16.0 g, 100 mmol) in AcOH (30 mL) dropwise over 20 min. at 10-15°C. The mixture was stirred at this temperature for 1 hour. Sn powder(12.4 g, 130 mmol) and H₂O (25 mL) were added and the mixture wasstirred at 80° C. for 1 hour. After an aqueous/EtOAc workup, the crudewas purified by column chromatography on silica gel (PE/EtOAc=5/1) andthen prep-HPLC (0.1% TFA as additive) to give3-bromonaphthalene-2,7-diol (8.2 g, yield 68%) as off-white solid.

Step 2: Synthesis of 6-bromo-7-(methoxymethoxy)naphthalen-2-ol: To asolution of the above product (4.00 g, 16.7 mmol) in MeCN (40 mL) wasadded K₂CO₃ (2.02 g, 14.5 mmol). The mixture was degassed for threetimes and MOMCl (1.87 g, 23.4 mmol) was added at −18° C. over 2 hoursvia syringe pump. The mixture was stirred at −18° C. for 2 hours andthen quenched with water (50 mL). The mixture was acidified with aqueousHCl (2M) until pH=6 and extracted with EtOAc (50 mL×3), washed withbrine (100 mL), dried over Na₂SO₄ and concentrated to give the crudeproduct, which was purified by column chromatography on silica gel(PE/EtOAc=10/1) to give the product (1.5 g, yield 32%) as off-whitesolid.

Step 3: Synthesis of Intermediate 11: To a solution of the above product(1.50 g, 5.30 mmol) in DMF (15 mL) was added K₂CO₃ (1.47 g, 10.6 mmol)and BnBr (1.18 g, 6.89 mmol). The mixture was stirred at 80° C.overnight. Water (5 mL) was added and the mixture was acidified withaqueous HCl (0.1M) until pH=7 carefully and extracted with EtOAc (30mL×3). The combined organic layers were washed with brine (50 mL), driedover Na₂SO₄ and concentrated in vacuo to give (1.98 g, yield 100%) asyellow solid.

Intermediate 12, 1-cyano-6-methoxynaphthalen-2-yltrifluoromethanesulfonate

Step 1: Synthesis of 1-bromo-6-methoxynaphthalen-2-ol: To a mixture of6-methoxynaphthalen-2-ol (20 g, 114.8 mmol) in DMF (250 mL) was added asolution of NBS (21.5 g, 120 mmol) in DMF (50 mL) over a 30 min period.The reaction mixture was stirred for 45 min and poured into water. Theprecipitate was collected by filtration and dried to give the desiredproduct (25.5 g, 87%) as white solid.

Step 2: Synthesis of 2-hydroxy-6-methoxy-1-naphthonitrile: A mixture ofthe above product (400 mg, 1.58 mol), Zn(CN)2 (742 mg, 6.32 mmol),Pd(PPh₃)₄ (913 mg, 0.79 mmol) in DMF (20 mL) was heated in a microwavereactor at 140° C. for 10 min. The reaction mixture was cooled andtreated with water (30 mL), extracted with EtOAc. The combined organiclayers were dried over MgSO₄, filtered and concentrated. The residue waspurified by silica gel chromatography (EA/PE=1/3) to give the product(240 mg, 76%) as white solid.

Step 3: Synthesis of Intermediate 12: To a solution of the above product(2.7 g, 13.5 mmol) and DIPEA (2.8 g, 27.1 mmol) in DCM (15 mL) at −78°C. was added trifluoromethanesulfonic anhydride (7.6 g, 27.1 mmol)dropwise. The mixture was allowed to slowly warm to 0° C. The resultingmixture was concentrated and purified by silica gel chromatography(EtOAc/PE=1/5) to give Intermediate 12 (4.0 g, 90%) as white solid. MS(ESI): m/z 332 [M+1]⁺.

Intermediate 13: 6-bromo-1-fluoronaphthalen-2-ol

To a solution of 6-bromonaphthalen-2-ol (2.23 g, 10.0 mmol) in CH₃CN (50mL) was added Selectfluor (4.20 g, 12.0 mmol). The reaction mixture washeated at 60° C. overnight and concentrated. The residue was partitionedbetween water (50 mL) and EtOAc (100 mL). The aqueous phase wasseparated and extracted with ethyl acetate. The combined organic layerswere washed with brine, dried over Na₂SO₄, filtered and concentrated.The residue was purified by silica gel chromatography to give6-bromo-1-fluoronaphthalen-2-ol using ethyl acetate and hexanes assolvents (2.0 g, 83% yield).

Example 35 GSNOR Assays

Various compounds were tested in vitro for their ability to inhibitGSNOR activity. GSNOR inhibitor compounds in Examples 1-33 had an IC₅₀of about <5 μM. GSNOR inhibitor compounds in Examples 1-3, 5, 6, 12, 15,17, 18, 20-33 had an IC₅₀ of about <0.1 μM. GSNOR inhibitor compounds inExamples 1, 2, 6, 12, 15, 17, 21-23, 25, 27-32 had an IC₅₀ of about<0.05 μM.

GSNOR expression and purification is described in Biochemistry 2000, 39,10720-10729.

GSNOR fermentation: Pre-cultures were grown from stabs of a GSNORglycerol stock in 2XYT media containing 100 μg/ml ampicillin after anovernight incubation at 37° C. Cells were then added to fresh 2XYT (4L)containing ampicillin and grown to an OD (A₆₀₀) of 0.6-0.9 at 37° C.before induction. GSNOR expression was induced with 0.1% arabinose in anovernight incubation at 20° C.

GSNOR Purification: E. coli cell paste was lysed by nitrogen cavitationand the clarified lysate purified by Ni affinity chromatography on anAKTA FPLC (Amersham Pharmacia). The column was eluted in 20 mM Tris pH8.0/250 mM NaCl with a 0-500 mM imidazole gradient. Eluted GSNORfractions containing the Smt-GSNOR fusion were digested overnight withUlp-1 at 4° C. to remove the affinity tag then re-run on the Ni columnunder the same conditions. GSNOR was recovered in the flowthroughfraction and for crystallography is further purified by Q-Sepharose andHeparin flowthrough chromatography in 20 mM Tris pH 8.0, 1 mM DTT, 10 uMZnSO₄.

GSNOR assay: GSNO and enzyme/NADH Solutions are made up fresh each day.The solutions are filtered and allowed to warm to room temperature. GSNOsolution: 100 mM NaPO₄ (pH 7.4), 0.480 mM GSNO. 396 μL of GSNO Solutionis added to a cuvette followed by 8 μL of test compound in DMSO (or DMSOonly for full reaction control) and mixed with the pipette tip.Compounds to be tested are made up at a stock concentration of 10 mM in100% DMSO. 2 fold serial dilutions are done in 100% DMSO. 8 μL of eachdilution are added to an assay so that the final concentration of DMSOin the assay is 1%. The concentrations of compounds tested range from100 to 0.003 μM. Enzyme/NADH solution: 100 mM NaPO₄ (pH 7.4), 0.600 mMNADH, 1.0 μg/mL GSNO Reductase. 396 μL of the Enzyme/NADH solution isadded to the cuvette to start the reaction. The cuvette is placed in theCary 3E UV/Visible Spectrophotometer and the change in 340 nmabsorbance/min at 25° C. is recorded for 3 minutes. The assays are donein triplicate for each compound concentration. IC₅₀'s for each compoundare calculated using the standard curve analysis in the Enzyme KineticsModule of SigmaPlot.

Final assay conditions: 100 mM NaPO₄, pH 7.4, 0.240 mM GSNO, 0.300 mMNADH, 0.5 μg/mL GSNO Reductase, and 1% DMSO. Final volume: 800μL/cuvette.

Example 36 Efficacy of GSNORi in Experimental Asthma

Experimental Asthma Model:

A mouse model of ovalbumin (OVA)-induced asthma was used to screen GSNORinhibitors for efficacy against methacholine (MCh)-inducedbronchoconstriction/airway hyper-responsiveness. This is a widely usedand well characterized model that presents with an acute, allergicasthma phenotype with similarities to human asthma. Efficacy of GSNORinhibitors was assessed using a protocol in which GSNOR inhibitors wereadministered after OVA sensitization and airway challenge, and prior tochallenge with MCh. Bronchoconstriction in response to challenge withincreasing doses of MCh was assessed using whole body plethysmography(P_(enh); Buxco). The amount of eosinophil infiltrate into thebronchoaveolar lavage fluid (BALF) was also determined as a measure oflung inflammation. The effects of GSNOR inhibitors were compared tovehicles and to Combivent (inhaled; IH) as the positive control.

Materials and Method

Allergen Sensitization and Challenge Protocol

OVA (500 μg/ml) in PBS was mixed with equal volumes of 10% (w/v)aluminum potassium sulfate in distilled water and incubated for 60 min.at room temperature after adjustment to pH 6.5 using 10 N NaOH. Aftercentrifugation at 750×g for 5 min, the OVA/alum pellet was resuspendedto the original volume in distilled water. Mice received anintraperitoneal (IP) injection of 100 μg OVA (0.2 mL of 500 μg/mL innormal saline) complexed with alum on day 0. Mice were anesthetized byIP injection of a 0.2-mL mixture of ketamine and xylazine (0.44 and 6.3mg/mL, respectively) in normal saline and were placed on a board in thesupine position. Two hundred fifty micrograms (100 μl of a 2.5 mg/ml) ofOVA (on day 8) and 125 μg (50 μl of 2.5 mg/ml) OVA (on days 15, 18, and21) were placed on the back of the tongue of each animal.

Pulmonary Function Testing (Penh)

In vivo airway responsiveness to methacholine was measured 24 h afterthe last OVA challenge in conscious, freely moving, spontaneouslybreathing mice with whole body plethysmography using a Buxco chamber(Wilmington, N.C.). Mice were challenged with aerosolized saline orincreasing doses of methacholine (5, 20, and 50 mg/mL) generated by anultrasonic nebulizer for 2 min. The degree of bronchoconstriction wasexpressed as enhanced pause (P_(enh)), a calculated dimensionless value,which correlates with the measurement of airway resistance, impedance,and intrapleural pressure in the same mouse. Penh readings were takenand averaged for 4 min. after each nebulization challenge. P_(enh) iscalculated as follows: P_(enh)=[(T_(e)/T_(r)−1)×(PEF/PIF)], where T_(e)is expiration time, T_(r) is relaxation time, PEF is peak expiratoryflow, and PIF is peak inspiratory flow×0.67 coefficient. The time forthe box pressure to change from a maximum to a user-defined percentageof the maximum represents the relaxation time. The T_(r) measurementbegins at the maximum box pressure and ends at 40%.

Eosinophil Infiltrate in BALF

After measurement of airway hyper-reactivity, the mice wereexsanguinated by cardiac puncture, and then BALF was collected fromeither both lungs or from the right lung after tying off the left lungat the mainstem bronchus. Total BALF cells were counted from a 0.05 mLaliquot, and the remaining fluid was centrifuged at 200×g for 10 min at4° C. Cell pellets were resuspended in saline containing 10% BSA withsmears made on glass slides. Eosinophils were stained for 5 min. with0.05% aqueous eosin and 5% acetone in distilled water, rinsed withdistilled water, and counterstained with 0.07% methylene blue.Alternatively, eosinophils and other leukocytes were stained withDiffQuik.

GSNOR Inhibitors and Controls

GSNOR inhibitors were reconstituted in phosphate buffered saline (PBS),pH 7.4, or 0.5% w/v carboxy methylcellulose at concentrations rangingfrom 0.00005 to 3 mg/mL. GSNOR inhibitors were administered to mice (10mL/kg) as a single dose or multiple dose either intravenously (IV) ororally via gavage. Dosing was performed from 30 min. to 72 h prior toMCh challenge. Effects of GSNOR inhibitors were compared to vehicledosed in the same manner.

Combivent was used as the positive control in all studies. Combivent(Boehringer Ingelheim) was administered to the lung using the inhalerdevice supplied with the product, but adapted for administration tomice, using a pipet tip. Combivent was administered 48 h, 24 h, and 1 hprior to MCh challenge. Each puff (or dose) of Combivent provides a doseof 18 μg ipatropium bromide (IpBr) and 103 μg albuterol sulfate orapproximately 0.9 mg/kg IpBr and 5 mg/kg albuterol.

Statistical Analyses

Area under the curve values for Penn enh across baseline, saline, andincreasing doses of MCh challenge were calculated using GraphPad Prism5.0 (San Diego, Calif.) and expressed as a percent of the respective (IVor orally administered) vehicle control. Statistical differences amongtreatment groups and the respective vehicle control group within eachstudy were calculated using one-way ANOVA, Dunnetts or Bonferronipost-hoc tests or t-test (JMP 8.0, SAS Institute, Cary, N.C. orMicrosoft Excel). A p value of <0.05 among the treatment groups and therespective vehicle control group was considered significantly different.

Results

In the OVA model of asthma, the compound of Example 1 significantly(p<0.05) decreased eosinophil infiltration in BAL by 44% of vehiclecontrol when given via three oral doses of 10 mg/kg at 48 h, 24 h, and 1h prior to assessment.

Example 37 Mouse Pharmacokinetic (PK) Study

Experimental Model

The mouse was used to determine the pharmacokinetics of compounds of theinvention. This species is widely used to assess the bioavailability ofcompounds by administering both oral (PO) and intravenous (IV) testarticles. Efficacy of the compounds of the invention was compared byassessing plasma exposure in male BALB/c mice either via IV or POadministration at the times of peak activity.

Materials and Methods

IV Administration of Compounds of the Invention

Compounds of the invention were reconstituted in a phosphate bufferedsaline (PBS)/10% Solutol (HS 15) clear solution resulting in aconcentration of 0.2 mg/mL and administered to mice (2 mg/kg) as asingle IV dose. Animals were dosed via the lateral tail vein. Bloodsamples were collected at designated time points (0.083, 0.25, 0.5, 1,2, 4, 8, 16, 24 hours) by cardiac puncture under isoflurane anesthesia(up to 1 mL blood per animal). The blood was collected into tubescontaining Li-Heparin. The blood samples were kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmawas transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

PO Administration of Compounds of the Invention

The compounds of the invention were reconstituted in 40% PropyleneGlycol/40% Propylene Carbonate/20% of a 5% Sucrose clear solutionresulting in a concentration of 2 mg/mL and administered to mice (10mg/kg) as a single oral dose via gavage. Blood samples were collected at0.25, 0.5, 1, 2, 4, 8, 12, 16, 20 and 24 hours post dose by cardiacpuncture under isoflurane anesthesia. The blood was collected in tubescontaining Li-Heparin. The blood samples were kept on ice untilcentrifugation within approximately 30 minutes of collection. The plasmawas transferred into labeled polypropylene tubes and frozen at −70° C.until analyzed by LC/MS/MS.

LC/MS/MS Analysis

Plasma samples at each timepoint were analyzed using a LC-MS/MS with alower limit of quantification (LLOQ) of 1 ng/mL. Plasma was analyzed todetermine the amount of the compound of the invention in each sample andregression curves generated for each compounds of the invention in therelevant matrices.

WinNonlin analysis was used for calculating PK parameters for both theIV and PO administrations:

PK parameters for IV portion—AUC_(last); AUC_(INF); T1/2; Cl; Vss;C_(max); MRT

PK parameters for PO portion—AUC_(last); AUC_(INF); T1/2; C_(max); Cl,MRT.

In addition to the above PK parameters, bioavailability (% F) wascalculated.

Results

The compound of Example 1 and the compound of Example 2 were tested andboth had an oral bioavailability of greater than 40%.

Example 38 Efficacy of GSNOR Inhibitors in Experimental InflammatoryBowel Disease (IBD)

Overview of the Models:

Acute and chronic models of dextran sodium sulfate (DSS)-induced IBD inmice were used to explore efficacy of GSNORi against this disease. Acuteand chronic DSS-induced IBD are widely used and well characterizedmodels that induce pathological changes in the colon similar to thoseobserved in the human disease. In these models and in human disease,epithelial cells within the crypts of the colon are disrupted, leadingto dysfunction of the epithelial barrier and the ensuing tissueinflammation, edema, and ulceration. GSNORi therapy may benefit IBD byrestoring s-nitrosoglutathione (GSNO) levels, and thus prevent orreverse the epithelial barrier dysfunction.

Acute Prophylactic Model:

Experimental IBD was induced by administration of DSS in the drinkingwater of male C57Bl/6 mice (N=8 to 10 mice per group) for 6 consecutivedays. GSNORi was dosed orally at doses of 0.1 to 10 mg/kg/day for 10days starting two days prior to and continuing two days post DSSexposure. Two days post DSS exposure, the effect of GSNORi was assessedin a blinded fashion via endoscopy and histopathology using a five pointscale ranging from a score=0 (normal tissue) through a score=4(ulcerative tissue damage and marked pathological changes). Levels ofcirculating cytokines involved in inflammatory pathways were alsoassessed. The effect of GSNORi was compared to vehicle treated controls.The corticosteroid, prednisolone, was used as the positive control inthis study and was administered daily at 3 mg/kg/day via oral dosing.Naïve mice (N=5) were also assessed as a normal tissue control.

Chronic Treatment Model:

Experimental IBD was induced by administration of DSS in the drinkingwater of male C57Bl/6 mice (N=10 to 12 mice per group) for 6 consecutivedays. GSNORi was dosed orally at doses of 10 mg/kg/day for 14 daysstarting one day after cessation of DSS exposure. Efficacy of GSNORi wasassessed in a blinded fashion via endoscopy after 7 days and 14 days ofGSNORi dosing and via histopathology after 14 days of GSNORi dosingusing a five point scale ranging from a score=0 (normal tissue) througha score=4 (ulcerative tissue damage and marked pathological changes).Levels of circulating cytokines involved in inflammatory pathways werealso assessed. The effect of GSNORi was compared to vehicle treatedcontrols. The corticosteroid, prednisolone, was used as the positivecontrol in this study and was administered daily at 3 mg/kg/day via oraldosing. Naïve mice (N=5) were also assessed as a normal tissue control.

Results:

The compound of Example 1 attenuated colon injury in mouse models ofacute and chronic DSS-induced IBD. In the acute model, the percent ofmice presenting with severe colon injury scores via endoscopy orhistopathology assessment was decreased by 17% or 21%, respectively, ofvehicle control after oral treatment with the compound of Example 1 at 1mg/kg/day for 10 consecutive days using a prophylactic dosing regimen.The compound of Example 1 dosed orally at 10 mg/kg/day for 10 days,significantly (p<0.05) decreased the percent of mice presenting withsevere endoscopy scores by 72% of vehicle control. In the chronic model,the percent of mice presenting with severe colon injury scores viaendoscopy or histopathology assessment was decreased by 50% or 17%,respectively, of vehicle control after oral treatment with the compoundof Example 1 at 10 mg/kg/day for up to 14 consecutive days using atreatment dosing regimen.

The compound of Example 2 attenuated colon injury in a mouse model ofacute DSS-induced IBD. The percent of mice presenting with severe coloninjury scores via endoscopy or histopathology assessments was decreasedby 58% (p<0.05) or 26%, respectively, of vehicle control after oraltreatment with the compound of Example 2 at 10 mg/kg/day for 10consecutive days using a prophylactic dosing regimen.

Example 39 Efficacy of GSNOR Inhibitors in Experimental ChronicObstructive Pulmonary Disease (COPD).

Short Duration Cigarette Smoke COPD Models

The efficacy of GSNOR inhibitors was assessed in a mouse model ofchronic obstructive pulmonary disease (COPD) induced by short duration(4 days or 11 days) of exposure to cigarette smoke. Infiltration ofinflammatory cells into the bronchoalveolar lavage fluid (BALF) and BALFlevels of chemokines involved in inflammation and tissue turnover/repairwere measured to assess the influences of GSNOR inhibitors on some ofthe early events associated with the initiation and progression of COPD.

Overview of the Models:

Efficacy of GSNOR inhibitors against COPD was explored using acute (4day) and subchronic (11 day) models of cigarette smoke-induced COPD inmice. Exposure of animals to cigarette smoke provides a model of COPD inwhich injury is induced by the same causative agent as in human diseaseand in which injury exhibits similarities to the human disease,including airway obstruction, airspace enlargement, and involvement ofinflammatory responses in these pathologies. In animal models, changesin lung pathology are only evident after extended (several months)duration of exposure to cigarette smoke, thus making chronic modelsprohibitive as effective screening tools. More recently, modelsexploring inflammatory responses after short duration (2 weeks or less)of smoke exposure in mice have been utilized as tools for screeningefficacy and mechanisms of action of novel therapeutics against COPD.The key roles of inflammation in the initiation and progression of COPD,make these short duration models relevant for initial tests of efficacyof novel therapeutics.

Acute (4 day) smoke exposure model: Female C57Bl/6 mice (N=8 per group)were exposed to cigarette smoke using a whole body exposure chamber.Mice were exposed daily for 4 consecutive days to 4 cycles of smoke from6 sequential cigarettes (Kentucky 3R4F without filter) with a 30 minutesmoke free interval between cycles. GSNOR inhibitors were administereddaily via oral dosing at 10 mg/kg/day for 7 days starting 2 days priorto smoke exposure and continuing 1 day post-exposure. The effects ofGSNOR inhibitors were assessed by quantitating the numbers of totalcells, leukocytes, and leukocytes differentials in the BALF via lightmicroscopy and the levels of BALF chemokines via ELISA at approximately24 h after the last smoke exposure. The effect of GSNOR inhibitors werecompared to vehicle treated controls. The PDE4 inhibitor, roflumilast,was used as the positive control for the study. A group of naïve mice(N=8) was exposed to air and used as a negative control for the study.

Subchronic (11 day) smoke exposure model: Female C57Bl/6 mice (N=10 pergroup) were exposed to cigarette smoke generated from Marlboro 100cigarettes without filters. Exposure times were 25 min. on study day 1,35 min. on study day 2, and 45 min. on study days 3 to 11. GSNORinhibitors were administered one hour prior to smoke exposure on eachday. GSNOR inhibitors were dosed orally at 1 to 10 mg/kg/day for 11days. The effects of GSNOR inhibitors were assessed by quantitating thenumber of total cells, and leukocytes differentials in the BALF vialight microscopy at 24 h after the last exposure. The effect of GSNORinhibitors were compared to vehicle treated controls and expressed aspercent inhibition of the cigarette smoke induced increases in BALF cellnumbers. Roflumilast was used as the positive control for the study andwas dosed at 5 mg/kg/day. A group of naïve mice (N=10) was exposed toair and dosed with vehicle as a negative control for the study.

Results:

The compound of Example 1 inhibited the smoke-induced increase in totalcells (p<0.05), macrophages (p<0.05), neutrophils (p<0.05), andlymphocytes in BAL by 40%, 40%, 49%, and 41%, respectively, when dosedorally at 10 mg/kg/day for 11 days in the subchronic 11 day model. Theseeffects of the compound of Example 1 were comparable to those ofroflumilast.

Example 40 An Exploratory Mouse Study of Acetaminophen Toxicity

S-nitrosoglutathione reductase (GSNOR) inhibition has been previouslyshown in our hands to ameliorate the negative manifestations ofgastrointestinal injury in animal models. As an extension of theseobservations, the effects of S-nitrosoglutathione (GSNO) or GSNORinhibitors (GSNORi) on acetaminophen (ACAP) induced liver toxicity canbe evaluated in a mouse model of liver injury. Blood samples arecollected for liver function assays and tissue samples are collected atthe end of the study for histopathologic examination.

Materials and Methods

GSNORi, GSNO, acetaminophen (ACAP, Sigma) Vehicles (½ cc syringes fordosing), Isoflurane, 18 1 cc syringes w/26 g needles for bloodcollection, 90 serum separator tubes for clinical chemistry.

General Study Design: Animals (5/group) are acclimated for at least 3days prior to dosing. On Study Day 1, acetaminophen treatment (300 mg/kgPO) was given a single time=0 to fasted animals. Two hours later, GSNORi(10 mg/kg/dose) or GSNO (5 mg/kg/dose) are intravenously administered tothe treatment groups. GSNORi or GSNO are given at 24 and 48 hours-posttheir initial administration to the treatment groups. Mice are observedfor signs of clinical toxicity and blood was collected at 6, 24, and 72hours post-ACAP administration for liver function tests: Alkalinephosphatase (ALK); Alanine aminotransferase (ALT); Aspartateaminotransferase (AST); Gamma glutamyltransferase (GGT) and Totalbilirubin (TBILI). Livers are collected at 72 hours for histopathologicexamination.

Study Outline

Group Treatment Dose Drug Concentration N 1 ACAP PO 300 mg/kg 10 ml/kg 52 Saline PO  0 mg/kg 10 ml/kg 5 3 GSNORi IV  10 mg/kg  1 mg/mL 5 4 GSNOIV  5 mg/kg  1 mg/mL 5 5 GSNORi IV + ACAP  10 m/k/300 m/k  1 mg/mL 5 6GSNO IV + ACAP  5 m/k/300 m/k  1 mg/mL 5

Study Calendar:

Day-6 Receive mice and place in regular cages Day-1 Fast animalsovernight Day 0 Weigh, PO ACAP time = 0, time = 2 IV GSNO or GSNORibleed all groups at 6 hr post-ACAP Day 1 Weigh, bleed all groups for 24hr LFTs, IV GSNO or GSNORi Day 2 Weigh, IV GSNO or GSNORi Day 3 Bleedfor 72 hr LFTs, collect livers for weight and histology

Vehicle, GSNO and GSNORi Preparation

The vehicle control article is Phosphate Buffered Saline (PBS) (notcontaining calcium, potassium, or magnesium) adjusted to pH 7.4. Thevehicle components are weighed into a container on a tared balance andbrought to volume with purified water (w/v). The 10× stock solution ismixed using a magnetic stirrer, as necessary. Thereafter, the 10× stocksolution is diluted with deionized water at a ratio of 1:9 (v/v). GSNOis warmed to room temperature before preparation of solutions. Prior touse, the PBS solution is nitrogen sparged. 1 mg/mL GSNO solutions arekept cold (i.e., kept on an ice bath) and protected from light and usedwithin 4 hours of preparation. GSNORi Preparation, the 1 mg/mLconcentration is reconstituted in phosphate buffered saline (PBS), pH7.4. GSNORi is administered to mice (10 mL/kg) as a single (IV) dailydose. Dosing is performed 2 hours post-ACAP administration and then 26and 50 hours later. Effects of GSNO or GSNORi are compared to ACAP andsaline vehicle dosed in the same manner.

Calculations: Mean body weights, mean liver organ weights and clinicalpathology endpoints (+/−SD) with T-test and ANOVA (alpha=0.05)comparison to vehicle control group. The clinical pathology data areprepared as mean values unless the data are not normally distributed, inwhich case, median values can be presented with the minimum and maximumvalue range.

Example 41 An Exploratory Study to Assess the Anti NASH FibroticActivity of GSNORi in STAM Mice

S-nitrosoglutathione reductase (GSNOR) inhibition has been previouslyshown in our hands to ameliorate the negative manifestations ofgastrointestinal injury and ACAP injury in mouse models. As an extensionof these observations, the effects of GSNOR inhibitors (GSNORi) abilityto reverse fibrotic activity in nonalcoholic steatohepatitis(NASH)-induced liver disease is evaluated in STAM (signal transducingadaptor molecule) mice. In these mice sequential changes are seen fromliver steatosis to fibrosis within two weeks and there are closesimilarities to human NASH histopathology.

Materials and Methods

GSNORi, Telmisartan, Vehicles (½ cc syringes for dosing), Isoflurane, 181 cc syringes w/26 g needles for blood collection, 90 serum separatortubes for clinical chemistry.

General Study Design: Animals (6/group) are acclimated prior tobeginning the Study. At 4 weeks of age the animals are put on a diet,group 1 (normal mice) receives a normal diet while groups 2-4 (STAMmice) are put on a high fat diet for the duration of the Study. At StudyWeek 7 the mice begin oral daily dosing with GSNORi and are sacrificedat Study Week 9. Mice are observed for signs of clinical toxicity andblood/tissue is collected for liver analyses: Plasma triglycerides (TG);Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); GeneExpression: Timp-1, α-SMA, collagen 3, TNF-α and MCP-1 as well ashistopathologic examination using HE staining for (NAFLD) activity scoreand Sirius-red staining (fibrosis area).

Study Outline

Group Treatment Diet Dose Drug Concentration N 1 normal ND  0 mg/kg 0ml/kg 6 2 S TAM + vehicle HFD 10 mg/kg 1 mg/mL 6 3 STAM + GSNORi IV HFD10 mg/kg 1 mg/mL 6 4 STAM + Telmisarten HFD 10 mg/kg 1 mg/mL 6 ND:normal diet, HFD: high fat diet

Calculations: Mean body weights, mean liver organ weights and clinicalpathology endpoints (+/−SD) with T-test and ANOVA (alpha=0.05)comparison to vehicle control group. The clinical pathology data areprepared as mean values unless the data are not normally distributed, inwhich case, median values were presented with the minimum and maximumvalue range.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention.

What is claimed is:
 1. The compound of Formula 1:

wherein R₁ is selected from the group consisting of H, F, and Cl; R_(2a)and R_(2b) are independently selected from the group consisting of H, F,Cl, Br, Me, OCH₃, and cyano; R_(2c) is selected from the groupconsisting of H, F, Cl, Br, Me, and OCH₃; X is selected from the groupconsisting of

A is selected from the group consisting of

R₃ is selected from the group consisting of F, Cl, Br, CH₃, CF₃, OCH₃,cyano, N(CH₃)₂, and morpholino; n is selected from the group consistingof 0, 1, and 2; R₄ is selected from the group consisting of H, F, Cl,Br, CH₃, CF₃, OCH₃, cyano, N(CH₃)₂, and morpholino;

with the proviso that when X is and A is COOH, then at least one of R₁,R_(2a), R_(2b), R_(2c), and R₄ is not hydrogen, or n must be >0, and R₃when meta to naphthalene, cannot be CH₃.
 2. The compound of claim 1wherein X is selected from the group consisting of


3. The compound of claim 1 wherein X is


4. The compound of claim 3 wherein R_(2c) is hydrogen.
 5. The compoundof claim 3 where R_(2b) is hydrogen.
 6. The compound of claim 1 whereinthe compound is a compound of Formula 3


7. The compound of claim 6 wherein X is


8. The compound of claim 7 where A is COOH.
 9. The compound of claim 1wherein the compound is a compound of Formula 5


10. The compound of claim 9 wherein X is


11. The compound of claim 10 wherein A is COOH.
 12. Use of a compound ofthe Formula 1 as defined in claim 1 or a pharmaceutically acceptablesalt thereof as a GSNOR inhibitor.
 13. A pharmaceutical compositioncomprising a therapeutically effective amount of a compound according toclaim 1 together with a pharmaceutically accepted carrier or excipient.14. A method of treatment of a disease or condition which comprisesadministering a therapeutically effective amount of a compound ofFormula 1 as defined in claim 1 to a patient in need thereof wherein thedisease or condition is a pulmonary disorder.
 15. The method of claim 14wherein the disease or condition is selected from the group consistingof asthma, chronic obstructive pulmonary disorder (COPD) and cysticfibrosis.
 16. A method of treatment of a disease or condition whichcomprises administering a therapeutically effective amount of a compoundof Formula 1 as defined in claim 1 to a patient in need thereof whereinthe disease or condition is an inflammatory disease.
 17. The method ofclaim 16 wherein the disease or condition is inflammatory bowel disease(IBD).
 18. A method of making a pharmaceutical composition comprisingcombining a compound of Formula 1 as defined in claim 1 with apharmaceutically acceptable carrier or excipient.